2 TMS K+ and water channel (conducts K+ (KD = 8 mM); blocked by Na+ (190 mM) (Renart et al., 2006) and tetrabutylammonium (Iwamoto et al., 2006)). Ion permeation occurs by ion-ion contacts in single file fashion through the selectivity filter (Köpfer et al. 2014). A narrow pore lined with four arrays of carbonyl groups is responsible for ion selectivity, whereas a conformational change of the four inner transmembrane helices (TMS2) is involved in gating (Baker et al. 2007). Two gates have been identified; one is located at the inner bundle crossing and is activated by H+ while the second gate is in the selectivity filter (Rauh et al. 2017). The C-terminal domain mediates pH modulation (Hirano et al., 2011; Pau et al., 2007). KcsA exhibits a global twisting motion upon gating (Shimizu et al., 2008). Activity is influenced by the phase of the lipid bilayer (Seeger et al. 2010), and occupancy of nonannular lipid binding sites increases the stability of the tetrameric complex (Triano et al. 2010). The open conformation of KcsA can disturb the bilayer integrity and catalyze the flipping of phospholipids (Nakao et al. 2014). This protein is identical to the KcsA orthologue (P0A333) in Streptomyces coelicolor. The stability of the pre domain in KcsA is stabilized by GCN4 (Yuchi et al. 2008). The potential role of pore hydration in channel gating has been evaluated (Blasic et al. 2015). Having multiple K+ ions bound simultaneously is required for selective K+ conduction, and a reduction in the number of bound K+ ions destroys the multi-ion selectivity mechanism utilized by K+ channels (Medovoy et al. 2016). The channel accomodates K+ and H2O molecules alternately in a K+-H2O-K+-H2O series through the channel (Kratochvil et al. 2016). Insertion of KcsA is spontaneous and directional as the cytosolic part of the protein does not translocate across the membrane barrier. Charged residues, not hydrophobic residues, are crucial for insertion of the unfolded protein into the membrane via electrostatic interactions between membrane and protein. A two-step mechanism was proposed. An initial electrostatic attraction between membrane and protein represents the first step prior to insertion of hydrophobic residues into the hydrocarbon core of the membrane (Altrichter et al. 2016). Bend, splay, and twist distinguish KcsA gate opening, filter opening, and filter-gate coupling, respectively (Mitchell and Leibler 2017). Details of the water permeability have been presented. Water flow through KcsA is halved by 200 mM K+ in the aqueous solution, which indicates an effective K+ dissociation constant in that range for a singly occupied channel. (Hoomann et al. 2013). A parameterized MARTINI program can be used to predict the hinging motions of the protein (Li et al. 2019). Activation of KcsA is initiated by proton binding to the pH gate upon an intracellular drop in pH which prompts a conformational switch, leading to a loss of affinity for potassium ions at the selectivity filter and therefore to channel inactivation (Rivera-Torres et al. 2016).
Voltage-sensitive Na+ channel, NaV1.7 (Cox et al., 2006). The human orthologue, SCN3A or Nav1.3, when mutated causes cryptogenic pediatric partial epilepsy (Holland et al., 2008; Zaman et al. 2020). Batrachotoxin (BTX) is a steroidal alkaloid neurotoxin that activates NaV channels through interacting with transmembrane domain-I-segment 6 (IS6) of these channels. Ginsenoside inhibits BTX binding (Lee et al. 2008). VGSCs are heterotrimeric complexes consisting of a single pore-forming alpha subunit joined by two beta subunits, a noncovalently linked beta1 or beta3 and a covalently linked beta2 or beta4 subunit (Hull and Isom 2017). The binding mode and functional components of the analgesic-antitumour peptide from Buthus martensii Karsch to human voltage-gated sodium channel 1.7 have been characterized (Zhao et al. 2019).
Type 2 Na+ channel, SCN2A or NaV1.2, of 2,005 aas and 24 TMSs. Mutations give rise to epileptic encephalophathy, Ohtahara syndrome (Nakamura et al. 2013). They may also give rise to autism (ASD) (Tavassoli et al. 2014). This protein is orthologous to the rat Na+ channel, TC# 1.A.1.10.1 and very similar to the type 1 Na+ channel (1.A.1.10.7). NaV1.2 has a single pore-forming alpha-subunit and two transmembrane beta-subunits. Expressed primarily in the brain, NaV1.2 is critical for initiation and propagation of action potentials. Milliseconds after the pore opens, sodium influx is terminated by inactivation processes mediated by regulatory proteins including calmodulin (CaM). Both calcium-free (apo) CaM and calcium-saturated CaM bind tightly to an IQ motif in the C-terminal tail of the alpha-subunit. Thermodynamic studies and solution structure (2KXW) of a C-domain fragment of apo 13C,15N- CaM (CaMC) bound to an unlabeled peptide with the sequence of the rat NaV1.2 IQ motif showed that apo CaMC (a) was necessary and sufficient for binding, and (b) bound more favorably than calcium-saturated CaMC. CaMN apparently does not influence apo CaM binding to NaV1.2IQp (Mahling et al. 2017).
Ca2+-regulated heart Na+ channel, Nav1.5, SCN5A or INa channel of 2016 aas. The COOH terminus functions in the control of channel inactivation and in pathologies caused by inherited mutations that disrupt it (Glaaser et al., 2006); regulated by ProTx-II Toxin (Smith et al. 2007), telethonin, the titin cap protein (167aas; secreted protein; O15273) (Mazzone et al., 2008), and the Mog1 protein, a central component of the channel complex (Wu et al., 2008). Nav1.5, the principal Na+ channel in the heart, possesses an ankyrin binding site, and direct interaction with ankyrin-G is required for the expression of Nav1.5 at the cardiomyocyte cell surface (Bennett and Healy, 2008; Lowe et al., 2008). Mutations cause type 3 long QT syndrome and type 1 Brugada syndrome, two distinct heritable arrhythmia syndromes (Mazzone et al., 2008; Kapplinger et al. 2010; Wang et al. 2015). SCN5A mutations causing arrhythmic dilated cardiomyopathy, commonly localized to the voltage-sensing mechanism, and giving rise to gating pore currents (currents that go through the voltage sensor) have been identified (McNair et al., 2011; Moreau et al., 2014). Patients with Brugada syndrome are prone to develop ventricular tachyarrhythmias that may lead to syncope, cardiac arrest or sudden cardiac death (Sheikh and Ranjan 2014) and (Kapplinger et al. 2015). Mutations causing disease have been identified (Qureshi et al. 2015). These give rise to arrhythias and cardiomyopathies (Moreau et al. 2015). Mutations that cause relative resistance to slow inactivation have been identified (Chancey et al. 2007). Green tea catechins are potential anti-arrhythmics because of the significant effect of Epigallocatechin-3-Gallate (E3G) on cardiac sodium channelopathies that display a hyperexcitability phenotype (Boukhabza et al. 2016). A mutatioin, R367G, causes the familial cardiac conductioin disease (Yu et al. 2017). The C-terminal domain of calmodulin (CaM) binds to an IQ motif in the C-terminal tail of the alpha-subunit of all NaV isoforms, and contributes to calcium-dependent pore-gating in some (Isbell et al. 2018). Ventricular fibrillation in patients with Brugada syndrome (BrS) is often initiated by premature ventricular contractions, and the presence of SCN5A mutations increases the risk upon exposure to sodium channel blockers in patients with or without baseline type-1 ECG (Amin et al. 2018). A mutation (R367G) is associated with familial cardiac conduction disease (Yu et al. 2017). Among ranolazine, flecainide, and mexiletine, only mexiletine restored inactivation kinetics of the currents of the mutant protein, A1656D (Kim et al. 2019). Epigallocatechin-3-gallate (EGCG) is protective against cardiovascular disorders due in part to its action on multiple molecular pathways and transmembrane proteins, including the cardiac Nav1.5 channels (Amarouch et al. 2020).
Voltage-sensitive Na+ channel, type 9, α-subunit, Nav1.7 or SCN9A (orthologous to 1.A.1.10.1). Loss of function, resulting from point mutations, results in a channelopathy called Congenital Insensitivity to Pain (CIP) (He et al. 2018), that causes the congenital inability to experience pain (Cregg et al., 2010; Kleopa, 2011). An S241T mutation causes inherited erythromelalgia IEM; erythermalgia, an autosomal dominant neuropathy characterized by burning pain in the extremities in response to mild warmth (due to altered gating) (Lampert et al., 2006; Drenth and Waxman, 2007). Gain-of-function mutations in the Na(v)1.7 channel lead to DRG neuron hyperexcitability associated with severe pain, whereas loss of the Na(v)1.7 channel in patients leads to indifference to pain (Dib-Hajj et al., 2007). Blocked by 1-benzazepin-2-one (Kd = 1.6 nM) (Williams et al., 2007). Mutations in the Nav1.7 Na channel α-subunit give rise to familial pain syndromes called chronic non-paoxysmal neuropathic pain (Catterall et al., 2008; Fischer and Waxman, 2010; Dabby et al. 2011 ). It interacts with the sodium channel beta3 (Scn3b), rather than the beta1 subunit, as well as the collapsing-response mediator protein (Crmp2) through which the analgesic drug lacosamide regulates Nav1.7 current (Kanellopoulos et al. 2018). The R1488 variant is totally inactive (He et al. 2018). Nav1.7 is inhibited by knottins (see TC# 8.B.19.2) (Agwa et al. 2018). Nav1.7 interacts with the following proteins: syn3b (TC# 8.a.17.1.2; the β3 subunit), Crmp2, Syt2 (Q8N9I0) and Tmed10 (P49755), and it also regulates opioid receptor efficacy (Kanellopoulos et al. 2018). Mutations in TRPA1 and Nav1.7 to insensitivity to pain-promoting algogens such as capsaicin, acid, and allyl isothiocyanate (AITC), have been documented (Eigenbrod et al. 2019). Nav1.7 is associated with endometrial cancer (Liu et al. 2019) and fever-associated seizures or epilepsy (FASE) (Ding et al. 2019).
Muscle plasmalemma, voltage-gated, L-type dihydropyridine receptor Ca2+ channel, α-1 subunit (DHPR) (Ba2+ > Ca2+), Cav1.1, CACNA1S,CACH1 CDCN1, CACNL1A3 of 1873 aas in the human orthologue. Distinc voltage sensor domains control voltage sensitivity and kinetics of current activation (Tuluc et al. 2016). Rapid changes in the transmembrane potential are detected by the voltage-gated Ca2+ channel, dihydropyridine receptor (DHPR), embedded in the sarcolemma. DHPR transmits the contractile signal to another Ca2+ channel, the ryanodine receptor (RyR1), embedded in the membrane of the sarcoplasmic reticulum (SR), which releases a large amounts of Ca2+ from the SR that initiate muscle contraction (Shishmarev 2020).
Two pore Ca2+ > Na+, Li+ or K+ (non-selective for these three monovalen caions) channel protein of 733 aas and 12 TMSs, TPC1 (Guo et al. 2017). The crystal structure of this vacuolar two-pore channel, a homodimer, has been solved (Guo et al. 2015) (Kintzer and Stroud 2016). Activation requires both voltage and cytosolic Ca2+. Ca2+ binding to the cytosolic EF-hand domain triggers conformational changes coupled to the pair of pore-lining inner helices from the first 6-TMS domains, whereas membrane potential only activates the second voltage-sensing domain, the conformational changes of which are coupled to the pair of inner helices from the second 6-TMS domains. Luminal Ca2+ or Ba2+ modulates voltage activation by stabilizing the second voltage-sensing domain in the resting state and shift voltage activation towards more positive potentials. The basis for understanding ion permeation, channel activation, the location of voltage-sensing domains and regulatory ion-binding sites is partially explained by the 3-d structure (Kintzer and Stroud 2016). Only the second Shaker domain senses voltage (Jaślan et al. 2016). It has a selectivity filter that is passable by hydrated divalent cations (Demidchik et al. 2018).
Voltage-dependent P/Q-type Ca2+ channel subunit α1A, CACNA1A (CACH4; CACN3; CACNL1A4) of 2,505 aas. 90% identical to 1.A.1.11.8. Associated with four neurological phenotypes: familial and sporadic hemiplegic migraine type 1 (FHM1, SHM1), episodic ataxia type 2 (EA2), spinocerebellar ataxia type 6 (SCA6) and epileptic encephalopathu with nerve atrophy (Reinson et al. 2016). A gain of function mutation gave symptoms of congenital ataxia, abnormal eye movements and developmental delay with severe attacks of hemiplegic migraine (García Segarra et al. 2014). Mutations can cause F/SHM with high penitrance (Prontera et al. 2018).
The voltage-dependent L-type Ca2+ channel α-subunit-1C (L-type Cav1.2), CACNA1C (CACH2, CACN2, CACNL1A1, CCHL1A1) of 2221 aa. Mutations cause Timothy's syndrome, a disorder associated with autism (Splawski et al., 2006). The C-terminus of Cav1.2 encodes a transcription factor (Gomez-Ospina et al., 2006). Cav1.2 associates with the α-2, δ-1, β and γ subunits (Yang et al., 2011). The CRAC channel activator STIM1 binds and inhibits L-type voltage-gated calcium channel, Cav1.2 (Park et al., 2010). This channel appears to function as the molecular switch for synaptic transmission (Atlas 2013). Intramembrane signalling occurs with syntaxin 1A for catecholamine release in chromaffin cells (Bachnoff et al. 2013). miR-153 intron RNA is a negative regulator of both insulin and dopamine secretion through its effect on Cacna1c expression, suggesting that IA-2beta and miR-153 have opposite functional effects on the secretory pathway (Xu et al. 2015). Co-localizes with Syntaxin-1A in nano clusters at the plasma membrane (Sajman et al. 2017). It is a high voltage-activated Ca2+ channel in contrast to Cav3.3 which is a low voltage-activated Ca2+ channel (Sanchez-Sandoval et al. 2018). Nifedipine blocks and potentiates this and other L-type VIC Ca2+ channels (Wang et al. 2018). Cav1.2 is upregulated when STIM1 is deficient (Pascual-Caro et al. 2018). CaV1.2 regulates chondrogenesis during limb development (Atsuta et al. 2019).
6TMS K+ channel (Munsey et al. 2002; Kuo et al., 2003). The kch gene, the only known potassium channel gene in E. coli, has the property to express both full-length Kch and its cytosolic domain (RCK) due to a methionine at position 240. The RCK domains form an octameric ring structure and regulate the gating of the potassium channels after having bound certain ligands. Several different gating ring structures have been reported for the soluble RCK domains. The octameric structure of Kch may be composed of two tetrameric full-length proteins through RCK interaction (Kuang et al. 2013). The RCK domains face the solution, and an RCK octameric gating ring arrangement does not form under certain conditions (Kuang et al. 2015).
2 TMS ( P-loop) Ca2+-gated K+ channel, MthK (see Jiang et al., 2002 for the crystal structure, and Parfenova et al., 2006 for mutations affecting open probability). For the studies of ion permeation and Ca2+ blockage, see Derebe et al., 2011. (structures: 3LDD_A and 2OGU_A.). Voltage-dependent K+ channels including MthK which lacks a canonical voltage sensor can undergo a gating process known as C-type inactivation, which involves entry into a nonconducting state through conformational changes near the channel's selectivity filter (Thomson and Rothberg, 2010). C-type inactivation may involve movements of transmembrane voltage sensor domains. In the absence of Ca2+, a single structure in a closed state was observed by cryoEM that was highly flexible with large rocking motions of the gating ring and bending of pore-lining helices (Fan et al. 2020). In Ca2+-bound conditions, several open-inactivated conformations were present with the different channel conformations being distinguished by rocking of the gating rings with respect to the transmembrane region. In all conformations displaying open channel pores, the N-terminus of one subunit of the channel tetramer sticks into the pore and plugs it. Deletion of this N terminus led to non-inactivating channels with structures of open states without a pore plug, indicating that this N-terminal peptide is responsible for a ball-and-chain inactivation mechanism (Fan et al. 2020). Lipid-protein interactions influence the conformational equilibrium between two states of the channel that differ according to whether a TMS has a kink. Two key residues in the kink region mediate crosstalk between two gates at the selectivity filter and the central cavity, respectively. Opening of one gate eventually leads to closure of the other (Gu and de Groot 2020).
Voltage-gated Na+ channel, NavCh or NavAb. The 3d-structure is known (3ROW; Payandeh et al., 2011; 4MW3A-D; 4+ selectivity through partial dehydration of Na+ via its direct interaction with conserved glutamate side chains). The pore is preferentially occupied by two ions, which can switch between different configurations by crossing low free-energy barriers (Furini and Domene, 2012). Jiang et al. 2018 presented high-resolution structures of NavAb with the analogous gating-charge mutations that have similar functional effects as in the human channels that cause hypokalaemic and normokalaemic periodic paralysis. Wisedchaisri et al. 2019 presented a cryo-EM structure of the resting state and a complete voltage-dependent gating mechanism via the voltage sensor (VS). The S4 segment of the VS is drawn intracellularly, with three gating charges passing through the transmembrane electric field. This movement forms an elbow connecting S4 to the S4-S5 linker, tightens the collar around the S6 activation gate, and prevents its opening. This structure supports the classical "sliding helix" mechanism of voltage sensing and provides a complete gating mechanism for voltage sensor function, pore opening, and activation-gate closure based on high-resolution structures of a single sodium channel protein (Wisedchaisri et al. 2019).
(see also TC#s 1.A.1.10.4 and 1.A.1.11.32).
The transport reaction catalyzed by NavCh is:
Na+ (in) ⇌ Na+ (out)
Bacterial voltage-gated sodium channel, Nav. 3-d crystal structures of vaious conformations are known (4P_3A A-D; 4PA7_A-D; 4P9P_A-D. etc.) (McCusker et al. 2012). It has its internal cavity accessible to the cytoplasmic surface as a result of a bend/rotation about a central residue in the carboxy-terminal TMS that opens the gate to allow entry of hydrated sodium ions. The molecular dynamics of ion transport through the open conformation has been analyzed (Ulmschneider et al. 2013). The C-terminal four helix coiled coil bundle domain couples inactivation with channel opening, depedent on the negatively charged linker region (Bagnéris et al. 2013). A NaVSp1-specific S4-S5 linker peptide induced both an increase in NaVSp1 current density and a negative shift in the activation curve, consistent with the S4-S5 linker stabilizing the open state (Malak et al. 2020).
Tetrameric 6 TMS subunit Na+ channel protein, NaV. Two low resolution cyroEM structures revealed two conformations reconstituted in lipid bilayers (Tsai et al. 2013). Despite a voltage sensor arrangement identical with that in the activated form, Tsai et al. 2013 observed two distinct pore domain structures: a prominent form with a relatively open inner gate, and a closed inner-gate conformation similar to the first prokaryotic Nav structure. Structural differences, together with mutational and electrophysiological analyses, indicated that widening of the inner gate was dependent on interactions among the S4-S5 linker, the N-terminal part of S5 and its adjoining part in S6, and on interhelical repulsion by a negatively charged C-terminal region subsequent to S6 (Tsai et al. 2013).
6 TMS cell volume sensitive, voltage-gated K+ channel, KCNQ4 or Kv7.4 (mutations cause DFNA2, an autosomal dominant form of progressive hearing loss) (forms homomers or heteromers with KCNQ3) (localized to the basal membrane of cochlear outer hair cells and in several nuclei of the central auditory pathway in the brainstem). Four splice variants form heterotetramers; each subunit has different voltage and calmodulin-sensitivities (Xu et al., 2007). Autosomal dominant mutant forms leading to progressive hearing loss (DFNA2) have been characterized (Kim et al. 2011). Phosphatidylinositol 4,5-bisphosphate (PIP2) and polyunsaturated fatty acids (PUFAs) impact ion channel function (Taylor and Sanders 2016). This channel may be present in mitochondria (Parrasia et al. 2019).
K+ voltage-gated channel, LQT-like subfamily; Kv7.1; KvLQT1. KCNQ1 (regulated by KCNE peptides (TC# 8.A.58) affect voltage sensor equilibrium (Rocheleau and Kobertz, 2007). Almost 300 mutations of KCNQ1 have been identified in patients, and most are linked to the long QT syndrome (LQT1), some in the voltage sensor (Peroz et al., 2008; Eldstrom et al. 2010; Qureshi et al. 2013; Ikrar et al. 2008). KCNQ1-KCNE1 complexes may interact intermittently with the actin cytoskeleton via the C-terminal region (Mashanov et al., 2010). The stoichiometry of the KCNQ1 - KCNE1 complex is flexible, with up to four KCNE1 subunits associating with the four KCNQ1 subunits of the channel (Nakajo et al., 2010). A familial mutation in the voltage-sensor of the KCNQ1 channel results in a cardiac phenotype (Henrion et al., 2012). KCNQ1 regulates insulin secretion in the MIN6 beta-cell line (Yamagata et al., 2011; Gofman et al., 2012). Electrostatic interactions of S4 arginines with E1 and S2 contribute to gating movements of S4, but coupling requires the lipid phosphatidylinositol 4,5-bisphosphate (PIP2) as voltage-sensing domain activation failed to open the pore in the absence of PIP2 (Zaydman et al. 2013). The D242N mutation causes impaired action potential adaptation to exercise and an increase in heart rate. Moreover, the D242 amino acylposition is involved in the KCNE1-mediated regulation of the voltage dependence of activation of the KV7.1 channel (Moreno et al. 2017). The KCNQ1 channel interacts with MinK (KCNE1) to cause pore constriction, generating the slow delayed rectifier (IKs) current in the heart (Jalily Hasani et al. 2018). KCNQ1 rescues TMC1 plasma membrane expression but not mechanosensitive channel activity (Harkcom et al. 2019). Activation of the neuronal Kv7/KCNQ/M-current represents an attractive therapeutic strategy for treatment of hyperexcitability-related neuropsychiatric disorders such as epilepsy, pain, and depression, and channel openers for treatment of antiepilepsy have been developed (Zhang et al. 2019). The relationship between mutation locations in KCNQ1, which is a major gene in long QT syndrome (LQTS), and phenotype has been analyzed and used for risk stratification (Yagi et al. 2018). The proximal C-terminal regions of KCNQ1 and KCNE1 participate in a physical and functional interaction during channel opening that is sensitive to perturbation (Chen et al. 2019). Retigabine analogs are activators of Kv7 channels (Ostacolo et al. 2020). People with borderline QTc prolongations were carriers of KCNQ1 mutations in TMSs 2 and 5, leading to haploinsufficiency, and they are potentially at risk of developing drug-induced arrhythmia (Gouas et al. 2004).
The archaeal voltage-regulated K channel, KvAP (Ruta et al., 2003). X-ray and solution structures are available. The latter shows phospholipid interactions with the isolated voltage sensor domain (Butterwick and MacKinnon 2010; Li et al. 2014). The gating-charge arginine in TMS4 of the voltage sensor forms part of the helical hairpin "paddle", and it moves 15-20 Å through the membrane to open the pore (Ruta et al., 2005). The orientation and depth of insertion of the voltage-sensing S4 helix has been determined (Doherty et al., 2010). A synthetic S6 segment derived from the KvAP channel self-assembles, permeabilizes lipid vesicles, and exhibits ion channel activity in bilayer lipid membrane (Verma et al., 2011). Thus the gating mechanism combines structural rearrangements and electric-field remodeling ( Li et al. 2014). KvAP has been reconstituted in Giant Unilamellar Vesicles (GUVs) (Garten et al. 2015). TMS4 (S4) which senses voltage also promotes membrane insertion of the voltage-sensor domain (Mishima et al. 2016). KvAP has a configuration consistent with a water channel, possibly underlying the conductance of protons, and other cations, through voltage-sensor domains (Freites et al. 2006). The structural dynamics of the paddle motif loop in the activated conformation of the KvAP voltage sensor have been studied from biophysical standpoints (Das et al. 2019).
|1.A.1.17.2||Voltage-gated K+ channel, Kv (Santos et al., 2008).||
Voltage-gated K+ channel, chain A, Shaker-related, Kv1.2 (Crystal structure known, Long et al., 2007; Chen et al. 2010). Functions with the auxiliary subunit, Ivβ1.2; 8.A.5.1.1) (Peters et al. 2009). Delemotte et al. (2010) described the effects of sensor domain mutations on molecular dynamics of Kv1.2. The Sigma 1 receptor (Q99720; Sigma non-opioid intracellular receptor 1) interacts with Kv1.2 to shape neuronal and behavioral responses to cocaine (Kourrich et al. 2013). Amino acid substitutions cause Shaker to become heat-sensing (opens with increasing temperature as for TrpV1) or cold-sensing (opens with decreasing temperature as for TrpM8) (Chowdhury et al. 2014). The Shaker Kv channel was truncated after the 4th transmembrane helix S4 (Shaker-iVSD) which showed altered gating kinetics and formed a cation-selective ion channel with a strong preference for protons (Zhao and Blunck 2016). Direct axon-to-myelin linkage by abundant KV1/Cx29 (TC# 1.A.24.1.12) channel interactions in rodent axons supports the idea of an electrically active role for myelin in increasing both the saltatory conduction velocity and the maximal propagation frequency in mammalian myelinated axons (Rash et al. 2016). A cryoEM structure (3 - 4 Å resolution; paddle chimeric channel; closed form) in nanodiscs has been determined (Matthies et al. 2018). Possible gating mechanisms have been discussed (Kariev and Green 2018; Infield et al. 2018).
Voltage-gated K+ channel, Kv1.1 or KCNA1. It is palmitoylated, modulating voltage sensing (Gubitosi-Klug et al. 2005). It is regulated by syntaxin (TC family 8.A.91) through dual action on channel surface expression and conductance (Feinshreiber et al., 2009). Defects cause episodic ataxia type 1 (EA1), an autosomal dominant K+ channelopathy accompanied by short attacks of cerebellar ataxia and dysarthria (D'Adamo et al. 2014). Direct axon-to-myelin linkage by abundant KV1/Cx29 channel interactions in rodent axons supports the idea of an electrically active role for myelin in increasing both the saltatory conduction velocity and the maximal propagation frequency in mammalian myelinated axons (Rash et al. 2016). Kv1.1 is present in bull sperm where it is necessary for normal sperm progressive motility, per cent capacitated spermatozoa (B-pattern) and the acrosome reaction (Gupta et al. 2018). Gating induces large aqueous volumetric remodeling (Díaz-Franulic et al. 2018). Paulhus et al. 2020 have reviewed the pathology of mutants in this protein and showed that epilepsy or seizure-related variants tend to cluster in the S1/S2 transmembrane domains and in the pore region of Kv1.1, whereas EA1-associated variants occur along the whole length of the protein.
The voltage-gated K+ channel subfamily D member 3, KCND3 or Kv4.3. Mutations cause spinocerebellar ataxia type 19 (Duarri et al. 2012). Positively charged residues in S4 contribute to channel inactivation and recovery (Skerritt and Campbell 2007). The crystal structure of Kv4.3 with its regulatory subunit, Kchip1, has been solved (2NZ0) (Wang et al. 2007).
Margatoxin-sensitive voltage-gated K+ channel, Kv1.3 (in plasma and mitochondrial membranes of T lymphocytes) (Szabò et al., 2005). Kv1.3 associates with the sequence similar (>80%) Kv1.5 protein in macrophage forming heteromers that like Kv1.3 homomers are r-margatoxin sensitive (Vicente et al., 2006). However, the heteromers have different biophysical and pharmacological properties. The Kv1.3 mitochondrial potassium channel is involved in apoptotic signalling in lymphocytes (Gulbins et al., 2010). Interactions between the C-terminus of Kv1.5 and Kvβ regulate pyridine nucleotide-dependent changes in channel gating (Tipparaju et al., 2012). Intracellular trafficking of the KV1.3 K+ channel is regulated by the pro-domain of a matrix metalloprotease (Nguyen et al. 2013). Direct binding of caveolin regulates Kv1 channels and allows association with lipid rafts (Pérez-Verdaguer et al. 2016). Addtionally, NavBeta1 interacts with the voltage sensing domain (VSD) of Kv1.3 through W172 in the transmembrane segment to modify the gating process (Kubota et al. 2017). During insertion of Kv1.3, the extended N-terminus of the second α-helix, S2, inside the ribosomal tunnel is converted into a helix in a transition that depends on the nascent peptide sequence at specific tunnel locations (Tu and Deutsch 2017). The microRNA, mmumiR449a, reduced the mRNA expression levels of transient receptor potential cation channel subfamily A member 1 (TRPA1), and calcium activated potassium channel subunit alpha1 (KCNMA1) and increased the level of transmembrane phosphatase with tension homology (TPTE) in the DRG cells (Lu et al. 2017). This channel is regulation by sterols (Balajthy et al. 2017). Loss of function causes atrial fibrillation, a rhythm disorder characterized by chaotic electrical activity of cardiac atria (Olson et al. 2006). The N-terminus and S1 of Kv1.5 can attract and coassemble with the rest of the channel (i.e. Frag(304-613)) to form a functional channel independently of the S1-S2 linkage (Lamothe et al. 2018). This channel may be present in mitochondria (Parrasia et al. 2019).
Voltage-gated K+ channel, Shaker. Shaker and Shab K+ channels are blocked by quinidine (Gomez-Lagunas, 2010). Also regulated by unsaturated fatty acids (Börjesson and Elinder, 2011). TMSs 3 and 4 comprise the voltage sensor paddle (Xu et al. 2013). Partially responsible for action potential repolarization during synaptic transmission (Ford and Davis 2014). Shaker K+ channels can be mutated in S4 to create an analogous "omega" pore (Held et al. 2018). The NMR structure of the isolated Shaker voltage-sensing domain in LPPG micelles has been reported (Chen et al. 2019). Substituting the first S4 arginine with a smaller amino acid opens a high-conductance pathway for solution cations in the Shaker K+ channel at rest. The cationic current does not flow through the central K+ pore and is influenced by mutation of a conserved residue in S2, suggesting that it flows through a protein pathway within the voltage-sensing domain (Tombola et al. 2005). The current can be carried by guanidinium ions, suggesting that this is the pathway for transmembrane arginine permeation. Tombola et al. 2005 proposed that when S4 moves, it ratchets between conformations in which one arginine after another occupies and occludes to ions in the narrowest part of this pathway.
K+ voltage-gated ether-a-go-go-related channel, H-ERG (KCNH2; Erg; HErg; Erg1, Kv11.1) subunit Kv11.1 (long QT syndrome type 2) (Gong et al., 2006; Chartrand et al. 2010; McBride et al. 2013). Selective expression of HERG and Kv2 channels influences proliferation of uterine cancer cells (Suzuki and Takimoto 2004). H-ERG forms a heteromeric K+ channel regulating cardiac repolarization, neuronal firing frequency and neoplastic cell growth (Szabó et al., 2011). Oligomerization is due to N-terminal interactions between two splice variants, hERG1a and hERG1b (Phartiyal et al., 2007). Structure function relationships of ERG channel activation and inhibition have been reviewed (Durdagi et al., 2010). Interactions between the N-terminal domain and the transmembrane core modulate hERG K channel gating (Fernández-Trillo et al., 2011). The marine algal toxin azaspiracid is an open state blocker (Twiner et al., 2012). Verapamil blocks channel activity by binding to Y652 and F656 in TMS S6 (Duan et al. 2007). Hydrophobic interactions between the voltage sensor and the channel domain mediate inactivation (Perry et al. 2013), but voltage sensing by the S4 segment can be transduced to the channel gate in the absence of physical continuity between the two modules (Lörinczi et al. 2015). Mutations give rise to long QT syndrome (Osterbur et al. 2015). Polyphenols such as caffeic acid, phenylethyl ester (CAPE) and curcumin inhibit by modification of gating, not by blocking the pore (Choi et al. 2013). Potassium ions can inhibit tumorigenesis through inducing apoptosis of hepatoma cells by upregulating potassium ion transport channel proteins HERG and VDAC1 (Xia et al. 2016). Incorrectly folded hERG can be degraded by Bag1-stimulated Trc-8-dependent proteolysis (Hantouche et al. 2016). The S1 helix regulates channel activity. Thus, S1 region mutations reduce both the action potential repolarizing current passed by Kv11.1 channels in cardiac myocytes, as well as the current passed in response to premature depolarizations that normally helps protect against the formation of ectopic beats (Phan et al. 2017). Interactions of beta1 integrins with hERG1 channels in cancer cells stimulate distinct signaling pathways that depended on the conformational state of hERG1 (Becchetti et al. 2017). ERG1 is sensitive to the alkaloid, ginsenoside 20(S) Rg3 which alters the gating of hERG1 channels by interacting with and stabilizing the voltage sensor domain in an activated state (Gardner et al. 2017). Channels split at the S4-S5 linker, at the intracellular S2-S3 loop, and at the extracellular S3-S4 loop are fully functional channel proteins (de la Peña et al. 2018). IKr is the rapidly activating component of the delayed rectifier potassium current, the ion current largely responsible for the repolarization of the cardiac action potential. Inherited forms of long QT syndrome (LQTS) in humans are linked to functional modifications in the Kv11.1 (hERG) ion channel and potentially life threatening arrhythmias. hERG1b affects the generation of the cardiac Ikr and plays an important role in cardiac electrophysiology (Perissinotti et al. 2018). X-ray crystallography and cryoEM have revealed features of the "nonswapped" transmembrane architecture, an "intrinsic ligand," and small hydrophobic pockets off a pore cavity. Drug block and inactivation mechanisms are discussed (Robertson and Morais-Cabral 2019). It forms a complex with β-integrin (TC#9.B.87.1.25) and NHE1 (TC# 2.A.36.1.13) (Iorio et al. 2020).
K+ voltage-gated channel, rEAG1; Kv10.1; rat ether a go-go channel 1 (962 aas). Blocked by Cs+, Ba2+ and quinidine (Schwarzer et al., 2008). Cysteines control the N- and C-linker-dependent gating of KCNH1 potassium channels (Sahoo et al., 2012). The 3-d structure has been determined at 3.8 Å resolution using single-particle cryo-EM with calmodulin bound. The structure suggests a novel mechanism of voltage-dependent gating. Calmodulin binding closes the potassium pore (Whicher and MacKinnon 2016). Eag1 has three intracellular domains: PAS, C-linker, and CNBHD. Whicher and MacKinnon 2019 demonstrated that the Eag1 intracellular domains are not required for voltage-dependent gating but likely interact with the VS to modulate gating. Specific interactions between the PAS, CNBHD, and VS domains modulate voltage-dependent gating, and VS movement destabilizes these interactions to promote channel opening. Mutations affecting these interactions render Eag1 insensitive to calmodulin inhibition (Whicher and MacKinnon 2019). The structure of the calmodulin insensitive mutant in a pre-open conformation suggests that channel opening may occur through a rotation of the intracellular domains, and calmodulin may prevent this rotation by stabilizing interactions between the VS and the other intracellular domains. Intracellular domains likely play a similar modulatory role in voltage-dependent gating of the related Kv11-12 channels. The human ortholog, EAG or EAG-1, is 989 aas long and is 95% identical to the rat protein. In ether-a-go-go K+ channels, voltage-dependent activation is modulated by ion binding to a site located in an extracellular-facing crevice between transmembrane segments S2 and S3 in the voltage sensor. Silverman et al. 2004 found that acidic residues, D278 in S2 and D327 in S3, are able to coordinate a variety of divalent cations, including Mg2+, Mn2+, and Ni2+, which have qualitatively similar functional effects, but different half-maximal effective concentrations. EAG (ether-a-go-go) voltage-dependent K+ channels with similarities and Differences in the structural organization and gating (Barros et al. 2020).
K+- and Na+-conducting NaK channel (3-D structure solves with Na+ and K+) (Shi et al., 2006). Exhibits tight structural and dynamic coupling between the selectivity filter and the channel scaffold (Brettmann et al. 2015).
The 6TMS bacterial cyclic nucleotide-regulated, voltage independent channel, MlotiK1 or MloK1 (Clayton et al., 2008). Gating involves large rearrangements of the cyclic nucleotide-binding domains (Mari et al., 2011). The electron crystalographic structure is available (PDB 4CHW)revealing ligand-induced structural changes (Schünke et al. 2011; Scherer et al. 2014; Kowal et al. 2014). Such changes may be lipid dependent (McCoy et al. 2014). High-speed atomic force microscopy has been used to measure millisecond to microsecond dynamics (Heath and Scheuring 2019).
Slo2.2 sodium-activated potassium channel subfamily T member 1 of 1217 aas and 6 putative N-terminal TMSs plus a P-loop. Cryo electron microscopic structures of the chicken orthologue at 4.5 Å resolution has been solved, revealing a large cytoplasmic gating ring in which resides the Na+-binding site and a transmembrane domain that closely resembles voltage-gated K+ channels (Hite et al. 2015).
Orthologue K+/Na+ pacemaker channel, Hcn4 (Scicchitano et al., 2012). Hyperpolarization-activated cyclic nucleotide-regulated HCN channels underlie the Na+-K+ permeable IH pacemaker current. As with other voltage-gated members of the 6-transmembrane KV channel superfamily, opening of HCN channels involves dilation of a helical bundle formed by the intracellular ends of S6, but this is promoted by inward, not outward, displacement of S4. Direct agonist binding to a ring of cyclic nucleotide-binding sites, one of which lies immediately distal to each S6 helix, imparts cAMP sensitivity to HCN channel opening. At depolarized potentials, HCN channels are further modulated by intracellular Mg2+ which blocks the open channel pore and blunts the inhibitory effect of outward K+ flux. Lyashchenko et al. 2014 showed that cAMP binding to the gating ring enhances not only channel opening but also the kinetics of Mg2+ block. Mutations in HCN4 cause sick sinus and the Brugada syndrome, cardiac abnormalities. Associated withfamiial sinus bradycardia (Boulton et al. 2017).
Hyperpolarization-activated cyclic nucleotide-gated (HCN) inward current carrying cationic channel, I(f), (HCN2/HCN4) (Ye and Nerbonne, 2009). Functional interactions between the HCN2 TM region and C-terminal region govern multiple CNB fold-mediated mechanisms, implying that the molecular mechanisms of autoinhibition, open-state trapping, and Quick-Activation include participation of TM region structures (Page et al. 2020).
Cyclic nucleotide-gated cation channel α3 (CNGA3 or CNG3); photoreceptor cGMP-gated channel α-subunit. Also possibly expressed in inner ear cell cells where it binds to an intracellular C-terminal domain of EMILIN1 (Selvakumar et al., 2012). Elastic network model analysis of the CNGA3 channel supports a modular model of allosteric gating, according to which protein domains are quasi-independent: they can move independently but are coupled to each other allosterically (Gofman et al. 2014).
|1.A.1.5.14||Probable cyclic nucleotide-gated ion channel 6 (AtCNGC6) (Cyclic nucleotide- and calmodulin-regulated ion channel 6)||
Hyperpolarization-activated and cyclic nucleotide-gated K+ channel, HCN (bCNG-1) (PNa+/PK+ ≈ 0.3). The human orthologue (O88703) is 863 aas in length and also catalyzes mixed monovalent cation currents K+:Na+= 4:1 (Lyashchenko and Tibbs et al., 2008). Biel et al. (2009) presented a detailed review of hyperpolarization-activated cation-channels. They are inhibited by nicotine and epibatidine which bind to the inner pore (Griguoli et al., 2010). They control cardiac and neuronal rhythmicity. HCN channels contain cyclic nucleotide-binding domains (CNBDs) in their C-terminal regions, linked to the pore-forming transmembrane segment with a C-linker. The C-linker couples the conformational changes caused by the direct binding of cyclic nucleotides to the HCN pore opening. Cyclic dinucleotides antagonize the effect of cyclic nucleotides in HCN4 but not in HCN2 channels. Interaction of the C-linker/CNBD with other parts of the channel appears to be necessary for cyclic-dinucleotide binding in HCN4 channels (Hayoz et al. 2017).
TWIK-1 (KCNK1, HOHO1, KCNO1) inward rectifier K+ channel (Enyedi and Czirják, 2010) expressed in the distal nephron segments (Orias et al. 1997). Lipid tails from both the upper and lower leaflets can partially penetrate into the pore (Aryal et al. 2015). The lipid tails do not sterically occlude the pore, but there is an inverse correlation between the presence of water within the hydrophobic barrier and the number of lipids tails within the lining of the pore (Aryal et al. 2015).
TREK-1 (KCNK2) K+ channel subunit (Regulated by group 1 metabotropic glutamate receptors and by diacylglycerols and phosphatidic acids) (Chemin et al., 2003). TREK-1, TREK-2 and TRAAK are all regulated by lysophosphatidic acid, converting these mechano-gated, pH voltage-sensitive channels into leak conductances (Chemin et al., 2005). The mammalian K2P2.1 potassium channel (TREK-1, KCNK2) is highly expressed in excitable tissues, where it plays a key role in the cellular mechanisms of neuroprotection, anaesthesia, pain perception and depression (Cohen et at., 2008). Alternative translation initiation in rat brain yields K2P2.1 potassium channels permeable to sodium (Thomas et al. 2008). The crystal structure of the human 2-pore domain K+ channel, K2P1 has been solved (Miller and Long, 2012). Multiple modalities converge on a common gate to control K2P channel function (Bagriantsev et al., 2011). TREK-1 mediates fast and slow glutamate release in astrocytes upon GPCR activation (Woo et al. 2012). It is a mechanosensitive K+ channel, present in rat bladder myocytes, which is activated by swelling and arachidonic acid (Fukasaku et al. 2016). The M2-hinges of TREK-1 and TREK-2 channels control their macroscopic current, subcellular localization and gating (Zhuo et al. 2017). The human ortholog has acc # O95069 and has an additional N-terminal 15 aas. BL-1249, a compound from the fenamate class of nonsteroidal anti-inflammatory drugs, is known to activate K2P2.1(TREK-1), the founding member of the thermo- and mechanosensitive TREK subfamily (Pope et al. 2018). Spadin and arachidonic acid, are known to suppress and activate TREK-1 channels, respectively (Pappa et al. 2020).
pH-dependent, voltage-insensitive, background potassium channel protein involved in maintaining the membrane potential, KCNK9, K2P9.1 or TASK3 (TASK-3) of 374 aas (Huang et al. 2011). Terbinafine is a selective activator of TASK3 (Wright et al. 2017). The response of the tandem pore potassium channel TASK-3 to voltage involves gating at the cytoplasmic mouth (Ashmole et al., 2009). TASK-3 is involved in several physiological and pathological processes including sleep/wake control, cognition and epilepsy (Tian et al. 2019). N-(2-((4-nitro-2-(trifluoromethyl)phenyl)amino)ethyl)benzamide (NPBA) is an activator (Tian et al. 2019). KCC2 regulates neuronal excitability and hippocampal activity via interaction with Task-3 channels (Goutierre et al. 2019). A biguanide compound, CHET3, is a highly selective allosteric activator, and TASK-3 is a druggable target for treating pain (Liao et al. 2019). This channel may be present in mitochondria (Parrasia et al. 2019).
Outward rectifying mechanosensitive 2-P (4 TMS) domain K+ channel, TREK-2 (KCNKA; KCNK10; K2P10). Activated by membrane stretch, acidic pH, arachidonic acid and unsaturated fatty acids. Dong et al. 2015 described crystal structures of the human TREK-2 channel (up to 3.4 angstrom resolution) in two conformations and in complex with norfluoxetine, the active metabolite of fluoxetine (Prozac) and a state-dependent blocker of TREK channels. Norfluoxetine binds within intramembrane fenestrations found in only one of these two conformations. Channel activation by arachidonic acid and mechanical stretch involves conversion between these states through movement of the pore-lining helices. This provides an explanation for TREK channel mechanosensitivity, regulation by diverse stimuli, and possible off-target effects of the serotonin reuptake inhibitor Prozac (Dong et al. 2015). The unique gating properties of TREK-2 and the mechanisms by which extracellular and intracellular stimuli harness pore gating allosterically have been studied (Zhuo et al. 2016). TREK-2 moves from the """"down"""" to """"up"""" conformation in direct response to membrane stretch. Aryal et al. 2017 showed how state-dependent interactions with lipids affect the movement of TREK-2, and how stretch influences both the inner pore and selectivity filter. They also demonstrated why direct pore block by lipid tails does not represent theprincipal mechanism of mechanogating (Aryal et al. 2017). The M2-hinges of TREK-1 and TREK-2 channels control their macroscopic current, subcellular localization and gating (Zhuo et al. 2017). TREK-2 responds to a diverse range of stimuli. Two states, termed """"up"""" and """"down,"""" are known from x-ray structural crystallographic studies and have been suggested to differ in conductance. Brennecke and de Groot 2018 found that the down state is less conductive than the up state. The introduction of membrane stretch in the simulations shifts the state of the channel toward an up configuration. Membrane pressure changes the conformation of the transmembrane helices directly and consequently also influences the channel conductance (Brennecke and de Groot 2018). 3-d structures are known (PDB 4XDJ_!-D. Phosphatidyl-(3,5)-bisphosphate (PI(3,5)P2) activates (Kirsch et al. 2018).
AMPA-selective glutamate ionotropic channel receptor (GIC; AMPAR), kainate-subtype, GluR-K1; GluR1; GluR-A; GluA1; Gria1 of 906 aas (preferentially monovalent cation selective). A mature complex contains GluR1, TARPs, and PSD-95 (Fukata et al. 2005). The receptor contributes to amygdala-dependent emotional learning and fear conditioning (Humeau et al., 2007). Transmembrane AMPAR regulatory protein (TARP) gamma-7 (TC#8.A.16.2.5) selectively enhances the synaptic expression of Ca2+-permeable (CP-AMPARs) and suppresses calcium-impermeable (CI-AMPAR) activities (Studniarczyk et al. 2013). Thus, TARPs modulate the pharmacology and gating of AMPA-type glutamate receptors (Soto et al. 2014). TARPs interact with the N-terminal domain of the AMPAR and control channel gating; residues in the receptor and the TARP involved in this interaction have been identified (Cais et al. 2014). The auxilary protein, Shisa9 or CKAMP44 (UniProt acc# B4DS77), has a C-terminal PDZ domain that allows interaction with scaffolding proteins and AMPA glutamate receptors (Karataeva et al. 2014). The transmembrane domain alone can tetramerize (Gan et al. 2016). The most potent and well-tolerated AMPA receptor inhibitors, used to treat epilepsy, act via a noncompetitive mechanism. The crystal structures of the rat AMPA-subtype GluA2 receptor in complex with three noncompetitive inhibitors have been solved. The inhibitors bind to a binding site, completely conserved between rat and human, at the interface between the ion channel and linkers connecting it to the ligand-binding domains (Yelshanskaya et al. 2016). The endogenous neuropeptide, cyclopropylglycine, at a physiological concentration of 1 μM, enhances the transmembrane AMPA currents in rat cerebellar Purkinje cells (Gudasheva et al. 2016). The energetics of glutamate binding have been estimated (Yu and Lau 2017). The TMEM108 protein (Q6UXF1 of 575 aas and 2 TMSs, N- and C-terminal, is required for surface expression of AMPA receptors (Jiao et al. 2017). CERC-611 is a selective antagonist of AMPA receptors containing transmembrane AMPA receptor regulatory protein (TARP; TC# 8.A.16) gamma-8 (Witkin et al. 2017). Drug effects, regulatory protein modulators and positive allosteric modulators have been reviewed (Fu et al. 2019). Herguedas et al. 2019 presented a cryo-EM structure of the heteromeric GluA1/2 receptor associated with two transmembrane AMPAR regulatory protein (TARP) gamma8 auxiliary subunits, the principal AMPAR complex at hippocampal synapses. The native heterotetrameric AMPA-R adopts various conformations, which reflect a variable separation of the two dimeric extracellular amino-terminal domains; members of the stargazin/TARP family of transmembrane proteins co-purify with AMPA-Rs and contribute to the density representing the transmembrane region of the complex. Glutamate and cyclothiazide altered the conformational equilibrium of the channel complex, suggesting that desensitization is related to separation of the N-terminal domains (Nakagawa et al. 2005). Positive allosteric modulators (PAMs) of AMPA receptors boost cognitive performance in clinical studies, and mibampator and BIIB104 discriminate between AMPARs complexed with distinct TARPs, and particularly those with lower stargazin/gamma2 efficacy such as BIIB104 (Ishii et al. 2020).
GriK2; GluK2; GluR6 glutamate receptor, ionotropic kainate 2. The 3-d structure is known (2XXY_A). The domain organization and function have been analyzed by Das et al. (2010). Two auxiliary subunits, Neto1 and Neto2 (Neuropilin and tolloid-like proteins) alter the trafficking, channel kinetics and pharmacology of the receptors (Howe 2014). They reduce inward rectification without altering calcium permeability (Fisher and Mott 2012). Interactions between the pore helix (M2) and adjacent segments of the transmembrane inner (M3) and outer (M1) helices may be involved in gating (Lopez et al. 2013). Mutations in the human GRIK2 (GLUR6) cause moderate-to-severe nonsyndromic autosomal recessive mental retardation (Motazacker et al. 2007). Kainate receptors regulate KCC2 (TC# 1.A.10.1.11) expression in the hippocampus (Pressey et al. 2017). GluR6, carrying the pore loop plus adjacent transmembrane domains of the prokaryotic, glutamate-gated, K+-selective GluR0 (TC# 1.A.10.2.1), adopted several electrophysiological properties of the donor pore uponpore transplantation (Hoffmann et al. 2006).
The NMDA receptor. The crystal structure of the N-terminal domains (GluN1 and GluN2) have been determined (PDB#3QEL; Talukder and Wollmuth, 2011). The ligand-specific deactivation time courses of GluN1/GluN2D NMDA receptors have been measured (Vance et al., 2011). NMDA receptors are Hebbian-like coincidence detectors, requiring binding of glycine and glutamate in combination with the relief of voltage-dependent magnesium block to open an ion conductive pore. Lee et al. 2014 presented X-ray structures of the Xenopus laevis GluN1-GluN2B NMDA receptor with the allosteric inhibitor, Ro25-6981, partial agonists and the ion channel blocker, MK-801. Receptor subunits are arranged in a 1-2-1-2 fashion, demonstrating extensive interactions between the amino-terminal and ligand-binding domains. The 3-TMS transmembrane domains harbour a closed-blocked ion channel, a pyramidal central vestibule lined by residues implicated in binding ion channel blockers and magnesium, and a approximately twofold symmetric arrangement of ion channel pore loops. GRIN2D mediates developmental and epileptic encephalopathy (XiangWei et al. 2019).
Glu2 AMPA receptor (GluR-2; GluR2-flop; CX614; GluA2). The 3-d structure is known at 3.6 Å resolution. It shows a 4-fold axis of symmetry in the transmembrane domain, and a 2-fold axis of symmetry overall, although it is a homotetramer (Sobolevsky et al. 2009). A structure showing an agoniar-bound form of the rat GluA2 receptor revealed conformational changes that occur during gating (Yelshanskaya et al. 2014). GluR2 interacts directly with β3 integrin (Pozo et al., 2012). In general, integrin receptors form macromolelcular complexes with ion channels (Becchetti et al. 2010). TARPS are required for AMP receptor function and trafficking, but seven other auxiliary subunits have also been identified (Sumioka 2013). For example, AMPA receptors are regulated by S-SCAM through TARPs (Danielson et al. 2012). The C-terminal domains of various TARPs (TC#8.A.16.2) play direct roles in the regulation of GluRs (Sager et al. 2011). Whole-genome analyses have linked multiple TARP loci to childhood epilepsy, schizophrenia and bipolar disorders (Kato et al. 2010). Thus, TARPs emerge as vital components of excitatory synapses that participate both in signal transduction and in neuropsychiatric disorders. The architecture of a fully occupied GluR2-TARP complex has been illucidated by cryoEM, showing the homomeric GluA2 AMPA receptor saturated with TARP Υ2 subunits, showing how the TARPs are arranged with four-fold symmetry around the ion channel domain, making extensive interactions with the M1, M2 and M4 TMSs (Zhao et al. 2016). The binding mode and sites for prototypical negative allosteric modulators at the GluA2 AMPA receptor revealing new details of the molecular basis of molulator binding and mechanisms of action (Stenum-Berg et al. 2019). Drug effects, regulatory protein modulators and positive allosteric modulators have been reviewed (Fu et al. 2019). TARP γ2 converts the desensitized state to the high-conductance state which exhibits tighter coupling between subunits in the extracellular parts of the receptor (Carrillo et al. 2020).
Glutamate receptor 4, GIC, AMPA-subtype, GluR4, GRIA4 or GluR-D (preferentially monovalent cation selective). Binding of the excitatory neurotransmitter, L-glutamate, induces a conformation change, leading to the opening of the cation channel, thereby converting the chemical signal to an electrical impulse. The receptor then desensitizes rapidly and enters a transient inactive state, characterized by the presence of bound agonist. In the presence of CACNG4, CACNG7 or CACNG8, GluR4 shows resensitization characterized by a delayed accumulation of current flux upon continued application of glutamate (Gill et al. 2008; Birdsey-Benson et al. 2010). De novo variants in GRIA4 lead to intellectual disability with or without seizures, gait abnormalities, problems of social behavior, and other variable features (Martin et al. 2017).
AMPA glutamate receptor 3 (GluR3, GluA3. GRIA3. LLUR3. GLURC) (non-selective monovalent cation channel and Ca2+ channel) (Ayalon et al., 2005; Midgett et al., 2012). Regulated by AMPA receptor regulatory proteins (TARPs) including stargazin and CNIH auxiliary subunits (Kim et al., 2010; Straub and Tomita, 2011; Jackson and Nicoll, 2011; Bats et al., 2012; Rigby et al. 2015). The domain architecture of a calcium-permeable AMPA receptor in a ligand-free conformation has been solved (Midgett et al., 2012). The TARP, stargazin, is elevated in the somatosensory cortex of Genetic Absence Epilepsy Rats (Kennard et al. 2011). TARPs alter the conformation of pore-forming subunits and thereby affect antagonist interactions (Cokić and Stein 2008). The structural basis of AMPAR regulation by TARP gamma2, or stargazin (STZ) involves variable interaction stoichiometries of the AMPAR-TARP complex, with one or two TARP molecules binding one tetrameric AMPAR (Twomey et al. 2016). The ion channel extracellular collar plays a role in gating and represents a hub for powerful allosteric modulation of AMPA receptor function (Yelshanskaya et al. 2017). The A653T mutation stabilizes the closed configuration of the channel and affects duration of sleep and awake periods in both humans and mice (Davies et al. 2017). The tetramer exhibits 4 distinct conductase leves due to independent subunit activation. Perampanel is an anticonvulsant drug that regulates gating (Yuan et al. 2019).
The homomeric cation channel/glutamate receptor/kainate 1, GluR5 (weakly responsive to glutamate) (expressed in the developing nervous system) (Bettler et al., 1990). The 3-d structures of the protein have been determined with agonists and antagonists. The agonist, domoic acid, stabilizes the ligand-binding core of the iGluR5 complex in a conformation that is 11 degrees more open than the conformation observed when the full agonist, (S)-glutamate, is bound (Hald et al. 2007). Kainate receptors regulate KCC2 expression in the hippocampus (Pressey et al. 2017). GluR5/ERK signaling is regulated by the phosphorylation and function of the glycine receptor alpha1ins subunit (TC# 9.A.14.3.4) in the spinal dorsal horn of mice (Zhang et al. 2019).
The heteromeric monovalent cation/Ca2+ channel/glutamate (NMDA) receptor NMDAR1/NMDAR2A/NMDAR2B/NMDAR2C) (Monyer et al., 1992). Note: NR2B is the same as NR3, GluN2A, GRIN2A or subunit epsilon (Schüler et al., 2008). Mediates voltage- and Mg2+-dependent control of Na+ and Ca2+ permeability (Yang et al., 2010). Mutations in the subunit, GRIN1, a 1464 aa protein, identified in patients with early-onset epileptic encephalopathy and profound developmental delay, are located in the transmembrane domain and the linker region between the ligand-binding and transmembrane domains (Yuan et al. 2014; Ohba et al. 2015). Karakas and Furukawa 2014 determined the crystal structure of the heterotetrameric GluN1-GluN2B NMDA receptor ion channel at 4 Å resolution. The receptor is arranged as a dimer of GluN1-GluN2B heterodimers with the twofold symmetry axis running through the entire molecule composed of an amino terminal domain, a ligand-binding domain, and a transmembrane domain. The GluN2 subunit regulates synaptic trafficking of AMPA in the neonatal mouse brain (Hamada et al. 2014). GRIN1 and GRIN2A mutations are associated with severe intellectual disability with cortical visual impairment, epilepsy and oculomotor and movement disorders being discriminating phenotypic features (Lemke et al. 2016; Chen et al. 2017).The cryoEM structure of a triheteromeric receptor including GluN1 (glycine binding), GluN2A and GluN2B (both glutamate binding) has been solved with and without a GluN2B allosteric antagonist, Ro 25-6981 (Lü et al. 2017). Ogden et al. 2017 implicated the pre-M1 region in gating, providing insight into how different subunits contribute to gating, and suggesting that mutations in the pre-M1 helix, such as those that cause epilepsy and developmental delays, can compromise neuronal health. The severity of GRIN2A (Glu2A)-related disorders can be predicted based on the positions of the mutations in the encoding gene (Strehlow et al. 2019). Knock-in mice expressing an ethanol-resistant GluN2A NMDA receptor subunit show altered responses to ethanol (Zamudio et al. 2019). Results of McDaniel et al. 2020 revealed the role of the pre-M1 helix in channel gating, implicated the surrounding amino acid environment in this mechanism, and suggested unique subunit-specific contributions of pre-M1 helices to GluN1 and GluN2 gating.
Glutamate-gated ionotropic K+ channel receptor, GluR0 (5TMSs). X-ray structures are available (PDB: 1IIT) (Lee et al. 2005; Lee et al. 2008) GluR6 (TC# 1.A.10.1.11), carrying the pore loop plus adjacent transmembrane domains of this prokaryotic, glutamate-gated, K+-selective GluR0, adopted several electrophysiological properties of the donor pore upon pore transplantation (Hoffmann et al. 2006).
Ammonia transporter and regulatory sensor, AmtB (Blauwkamp and Ninfa, 2003; Khademi et al., 2004). It has a cleavable N-terminal signal peptide, and while Amt proteins in Gram-negative bacteria appear to utilize a signal peptide, the homologous proteins in Gram-positive organisms do not (Thornton et al. 2006).
|1.A.11.3.2||High-affinity ammonia transporter and sensor, Mep2 (also an NH4+ sensor) (Javelle et al., 2003a; Rutherford et al., 2008) (has a pair of conserved his/his residues; mutation to his/glu as in Mep1 leads to uncoupling of transport and sensor functions (Boeckstaens et al., 2008))||
Rhesus (Rh) type C glycoprotein NH3/NH4+ transporter, RhCG (also called tumor-related protein DRC2) (Bakouh et al., 2004; Worrell et al., 2007). Zidi-Yahiaoui et al. (2009) have described characteristics of the pore/vestibule. The structure is known to 2.1 Å resolution (Gruswitz et al., 2010). Each monomer contains 12 transmembrane helices, one more than in the bacterial homologs. Reconstituted into proteoliposomes, RhCG conducts NH3 to raise the internal pH. Models of the erythrocyte Rh complex based on the RhCG structure suggest that the erythrocytic Rh complex is composed of stochastically assembled heterotrimers of RhAG, RhD, and RhCE (Gruswitz et al., 2010).
The RH50 NH3 channel (most like human Rh proteins TC# 1.A.11.4.1 and 2; 36-38% identity) (Cherif-Zahar et al., 2007). The Rh CO2 channel protein (3-D structure ± CO2 available) (3B9Z_A; 3B9Y_A) (Li et al., 2007; Lupo et al., 2007) (also transports methyl ammonia) (Weidinger et al., 2007).
Nuclear chloride channel-27, NCC27 or CLIC1 (Br- > Cl- > I-) (241 aas). CLIC1 has two charged residues, K37 and R29, in its single TMS which are important for the biophysical properties of the channel (Averaimo et al. 2013). A putative Lys37-Trp35 cation-pi interaction stabilizes the active dimeric form of the CLIC1 TMS in membranes (Peter et al. 2013). This channel may play a role in cancer (Peretti et al. 2014). A positively charged motif at the C-terminus of the single TMS enhances membrane partitioning and insertion via electrostatic contacts. It also functions as an electrostatic plug to anchor the TMS in membranes and is involved in orientating the TMS with respect to the cis and trans faces of the membrane (Peter et al. 2014). The CLIC1 protein accumulates in the circulating monocyte membrane during neurodegeneration (Carlini et al. 2020).
The Janus protein, CLIC2. The 3-D structure of its water soluble form has been determined at 1.8 Å resolution (Cromer et al., 2007). CLIC2 interacts with the skeletal ryanodine receptor (RyR1) and modulates its channel activity (Meng et al., 2009).
Chloride intracellular channel protein 4, CLIC4. Regulates the histamine H3 receptor (Maeda et al., 2008)) Discriminates poorly between anions and cations (Singh and Ashley, 2007). 76% identical to CLIC5; it may play a role in cancer (Peretti et al. 2014). CLIC5 was the first mitochondrial chloride channel to be identified in the inner mitochondrial membrane, while CLIC4 is located predominantly in the outer mitochondrial membrane. Gururaja Rao et al. 2020 discussed the intracellular chloride channels, their roles in pathologies, such as cardiovascular, cancer, and neurodegenerative diseases, and developments concerning their usage as theraputic targets in humans.
Intracellular Cl- channel-3 (CLIC3). The 3-d structure is known (3FY7). This protein is associated with pregnancy disorders (Murthi et al., 2012).
Putative Glutathione S-transferase. Pore formation has not been demonstrated in prokaryotes.
The 7 TMS proton-sensitive Ca2+ leak channel, YetJ. The activity and high resolution 3-d structure have been determined (Chang et al. 2014).
Formate uptake/efflux permease, FocA. It catalyzes bidirectional transport, has a pentameric aquaporin-like (TC# 1.A.8) structure, and may function by a channel-type mechanism (Falke et al., 2009; Wang et al. 2009). The structure at 2.25 Å resolution has been determined (Wang et al., 2009). The protein is encoded in an operon with pyruvate-formate lyase, PflB. A pyruvate:formate antiport mechanism has been suggested (Moraes and Reithmeier 2012). The C-terminal 6 aas are required for formate transport, but not for homopentamer formation (Hunger et al. 2017). The N-terminus of FocA interacts with PflB, and this interaction is essential for optimal formate translocation (Doberenz et al. 2014). In fact, the GREs, TdcE and PflB, interact with the FNT channel protein, probably to control formate translocation by FocA (Falke et al. 2016).
Nitrite uptake/efflux channel (Jia et al. 2009).
Hydrosulfide (hydrogen sulfide; HS-), Fnt3 (Hsc) channel. Also probably transports chloride, formate and nitrite. The 3-d crystal structure (2.2Å resolution in the closed state) is known (PDB# 3TE2) (Czyzewski and Wang, 2012). The Fnt3 gene is linked to the asrABC operon encoding the sulfite (SO32-) reductase that gives HS- as the product (Czyzewski and Wang 2012).
TMEM16 of 735 aas and 10 TMSs. Operates as a Ca2+-activated lipid scramblase (Wang et al. 2018). Each subunit of the homodimer contains a hydrophilic membrane-traversing cavity that is exposed to the lipid bilayer as a potential site of catalysis. This cavity harbours a conserved Ca2+-binding site, located within the hydrophobic core of the membrane. Mutations of residues involved in Ca2+ coordination affect both lipid scrambling in N. haematococca TMEM16 and ion conduction in the Cl- channel of TMEM16A. The structure reveals the general architecture of the family and its mode of Ca2+ activation (Brunner et al. 2014). While the cytoplasmic portion of the protein is important for function, it does not appear to regulate scramblase activity via a detectable conformational change (Andra et al. 2018). Dynamic modulation of the lipid translocation groove generates a conductive ion channel in Ca2+-bound nhTMEM16 (Khelashvili et al. 2019) (see family description).
Tic110 channel protein. The x-ray structure (4.2Å resolution) of Tic110B and C from Cyanidioschyzon merolae is known (Tsai et al., 2013). The C-terminal half of Tic110 posesses a rod-shaped helix-repeat structure that is too flattened and elongated to be a channel. The structure is most similar to the HEAT-repeat motif that functions as scaffolds for protein-protein interactions (Tsai et al., 2013).
Matrix protein, M2, an acid activated drug-sensitive proton channel. Transport involves binding to the four His-37s and transfer to water molecules on the inside of the channel (Acharya et al., 2010). Functional properties and structure are known (Hong and Degrado 2012). The cytoplasmic tail facilitates proton conduction (Liao et al. 2015). It is a dimer of dimers (Andreas et al. 2015). The four TMSs flanking the channel lumen alone seem to determine the proton conduction mechanism (Liang et al. 2016). His-37 forms a planar tetrad in the configuration of the bundle accepting and translocating the incoming protons from the N terminal side, exterior of the virus, to the C terminal side, inside the virus (Kalita and Fischer 2017). The cholesterol binding site in M2 that mediates membrane scission in a cholesterol-dependent manner to cause virus budding and release has been identified (Elkins et al. 2017).Transport-related conformational changes coupled to water and H+ movements have been studied (Mandala et al. 2018). The L46P mutant confers a novel allosteric mechanism of resistance towards the influenza A virus M2 S31N proton channel blockers (Musharrafieh et al. 2019). The C-terminal domain of M2 may serve as a sensor that regulates how M2 participates in critical events in the viral infection cycle (Kim et al. 2019).
G-protein-activated inward rectifying K+ channel, Kir3.2, KATP2, KCNJ7 or GIRK2 of 423 aas (Inanobe et al., 2011; Yokogawa et al. 2011). Important in regulating heart rate and neuronal excitability. Activated by binding of the βγ-subunit complex to the cytoplasmic pore gate (Yokogawa et al. 2011). Chen et al. 2017 found that the G-protein-gated inwardly rectifying K+ (GIRK) channel is activated by Ivermectin (IVM) directly. Cholesterol binds to and upregulates GIRK channels (GIRK2 and 4), and the binding sites have been determined (Rosenhouse-Dantsker 2018). An inherited gain-of-function mutation in the human GIRK3.4 causes familial human sinus node dysfunction (SND). The increased activity of GIRK channels is likely to lead to a sustained hyperpolarization of pacemaker cells and thereby reduces heart rate (Kuß et al. 2019). GIRK2 channels are abundantly expressed in the heart and require that phosphatidylinositol bisphosphate (PIP2) is present so that intracellular channel-gating regulators such as Gbetagamma (Gβγ)and Na+ ions maintain the channel-open state. Li et al. 2019 determined how each regulator uses the channel domain movements to control gate transitions. Na+ controls the cytosolic gate of the channel through an anti-clockwise rotation, whereas Gbetagamma stabilizes the transmembrane gate in the open state through a rocking movement of the cytosolic domain. Both effects altered the way by which the channel interacts with PIP2 and thereby stabilized the open states of the respective gates (Li et al. 2019).
Prokaryotic K+-selective Kir channel KirBac1.1 (selectivity: K+ = Rb+ = Cs+ >> Li+, Na+ or NMGM) (Enkvetchakul et al., 2004), inward rectifying (Cheng et al., 2009). Closure of the Kir1.1 pH gate results from steric occlusion of the permeation path by the convergence of four leucines (or phenylalanines) at the cytoplasmic apex of the inner transmembrane helices. In the open state, K+ crosses the pH gate together with its hydration shell (Sackin et al. 2005). The inhibitory cholesterol binding site has been identified (Fürst et al. 2014). Conformational changes associated with an open activation gate have been identified, and these suggest a unique allosteric pathway that ties the selectivity filter to the activation gate through interactions between both transmembrane helices, the turret, the selectivity filter loop, and the pore helix. Specific residues involved in this conformational exchange that are highly conserved among human Kir channels have also been identified (Amani et al. 2020). Anionic lipids, especially cardiolipin initiates a concerted rotation of the cytoplasmic domain subunits. This action buries ionic side chains away from the bulk water, while allowing water greater access to the K+ conduction pathway (Borcik et al. 2020).
The KirBac3.1 K+ channel (a dimer of dimers with gating visualized by atomic force microscopy, Jaroslawski et al., 2007) (regulated by binding lipids, G-proteins, nucleotides, and ions (H+, Ca2+, and Mg2+)). The 3-D structure is available (1XL6_A). The inhibitory cholesterol binding site has been identified (Fürst et al. 2014). The constricted opening in this, and presumably other, Kir channels does not impede potassium conduction (Black et al. 2020).
Inward rectifier potassium channel
|1.A.20.1.1||BNip3 channel-forming protein (Bocharov et al., 2007)||
Apoptosis regulator Bcl-X(L) of 233 aas. Also called Bcl2-like protein 1, isoform 1. Membrane insertion of the soluble form has been characterized (Vargas-Uribe et al. 2013). The cytosolic domain of Bcl-2 forms small pores in the mitochondrial outer membrane (Peng et al. 2009).
|1.A.21.1.12||The Cell Death (CED-9) protein (Siskind et al., 2008)||
The mitochondrial apoptosis-inducing channel-forming protein, BAX. The C-terminal helix mediates membrane binding and pore formation (Garg et al. 2012). BAX pores are large enough to allow cytochrome c release and it activates the mitochondrial permeabilty transition pore; both play a role in programmed cell death, but the latter is quantitatively more important (Gómez-Crisóstomo et al. 2013). Bax functions like a holin when expressed in bacteria (Pang et al. 2011). Bax (and likely Bak) dimers assemble into oligomers with an even number of molecules that fully or partially delineate pores of different sizes to permeabilize the mitochondrial outer membrane (MOM) during apoptosis (Cosentino and García-Sáez 2016). The membrane domain of Bax interacts with other members of the Bcl-2 family to form hetero-oligomers (Andreu-Fernández et al. 2017). Uren et al. 2017 reviewed how clusters of dimers and their lipid-mediated interactions provide a molecular explanation for the heterogeneous assemblies of Bak and Bax observed during apoptosis. After BAK/BAX activation and cytochrome c loss, the mitochondrial network breaks down, and large BAK/BAX pores appear in the outer membrane. These macropores allow the inner membrane an outlet through which it herniated, carrying with it mitochondrial matrix components including the mitochondrial genome (McArthur et al. 2018). The core/dimerization domain of Bax and Bak is water exposed with only helices 4 and 5 in membrane contact, whereas the piercing/latch domain is in peripheral membrane contact, with helix 9 being transmembrane (Bleicken et al. 2018). The mechanism of the membrane disruption and pore-formation by the BAX C-terminal TMS has been investigated (Jiang and Zhang 2019). Bax membrane permeabilization results from oligomerization of transmembrane monomers (Annis et al. 2005).
The mitochondrial apoptosis-inducing channel-forming protein, BAK. 3-D structures are known (2IMT_A). Functions like a holin when expressed in bacteria (Pang et al. 2011). Formation of the apoptotic pore involves a flexible C-terminal domain (Iyer et al. 2015). Bax (and likely Bak) dimers assemble into oligomers with an even number of molecules that fully or partially delineate pores of different sizes to permeabilize the mitochondrial outer membrane (MOM) during apoptosis (Cosentino and García-Sáez 2016). BAK is a C-tail-anchored mitochondrial outer membrane protein (Setoguchi et al. 2006). BAK plays a role in peroxisomal permeability, similar to mitochondrial outer membrane permeabilization (Hosoi et al. 2017). Uren et al. 2017 reviewed how clusters of dimers and their lipid-mediated interactions provide a molecular explanation for the heterogeneous assemblies of Bak and Bax observed during apoptosis. After BAK/BAX activation and cytochrome c loss, the mitochondrial network breaks down, and large BAK/BAX pores appear in the outer membrane. These macropores allow the inner membrane an outlet through which it herniates, carrying with it mitochondrial matrix components including the mitochondrial genome (McArthur et al. 2018).
Pro-survival Bcl-w protein. Binds the BH3-only protein Bop to inhibit Bop-induced apoptosis (Zhang et al. 2012). The structure is known (PDB# 1MK3).
Large mechanosensitive ion channel: MscL; catalyzes efflux of ions (slightly cation selective), osmolytes and small proteins. Protein-lipid interactions are important for gating, dependent on TMS tilting (Iscla et al., 2011b). The carboxyl-terminal cytoplasmic helices assemble into a pentameric bundle that resembles cartilage oligomeric matrix protein, and these are required for the selective formation of the pentamer (Ando et al. 2015). Lysophospholipids can increase the size of particles that can be transported (Foo et al. 2015). 500 - 700 channels are needed for 80% survival, a number of channels similar to that found in cell (Chure et al. 2018). its activation threshold decreases with membrane thickness; the membrane-thickness-dependent MscL opening mainly arises from structural changes in MscL to match the altered membrane thickness by stretching (Katsuta et al. 2018).
Large conductance mechanosensitive channel protein, MscL, of 101 aas and 2 TMSs. When the membrane is stretched, MscL responds to the increase of membrane tension and opens a nonselective pore to about 30 A wide, exhibiting a large unitary conductance of approximately 3 nS. The structures of this archaeal MscL, trapped in the closed and expanded intermediate states, has been solved (Li et al. 2015). The comparative analysis of these two new structures reveals significant conformational rearrangements in the different domains of MscL. The large changes observed in the tilt angles of the two transmembrane helices (TMS1 and TMS2) fit well with the helix-pivoting model. Meanwhile, the periplasmic loop region transforms from a folded structure, containing an omega-shaped loop and a short beta-hairpin, to an extended and partly disordered conformation during channel expansion. Moreover, a significant rotating and sliding of the N-terminal helix (N-helix) is coupled to the tilting movements of TMS1 and TMS2. The dynamic relationships between the N-helix and TMS1/TMS2 suggest that the N-helix serves as a membrane-anchored stopper that limits the tilts of TM1 and TM2 in the gating process (Li et al. 2015).
Large mechanosensitive ion channel of 151 aas and 2 TMSs. The 3-D structure is known, and it may reflect a nearly closed rather than fully closed state. Modeling support a clockwise rotation of the pore-forming first TMS promotes gating (Bartlett et al. 2004).
Major MscS channel protein, YggB. Seven residues, mostly hydrophobic, in the first and second transmembrane helices are lipid-sensing residues (Malcolm et al., 2011). X-ray structures are available (Lai et al. 2013). The cytoplasmic cage domain senses macromolecular crowding (Rowe et al. 2014). A gating mechanism has been proposed (Malcolm et al. 2015). The thermodynamics of K+ leak have been studied (Koprowski et al. 2015). In the MscS crystal structure (PDB 2OAU ), a narrow, hydrophobic opening is visible in the crystal structure, and a vapor lock, created by hydrophobic seals consisting of L105 and L109, is the barrier to water and ions (Rasmussen et al. 2015). The voltage dependence of inactivation occurs independently of the positive charges of R46, R54, and R74 (Nomura et al. 2016). The closed-to-open transition may involve rotation and tilt of the pore-lining helices (Edwards et al. 2005).
Connexin 43 (gap junction α-1 protein), CX43 encoded by the GJA1 gene (transports ATP, ADP and AMP better than CX32 does; Goldberg et al., 2002). Hemichannels mediate efflux of glutathione, glutamate and other amino acids as well as ATP (Stridh et al., 2008; Kang et al., 2008). CX43 has a half life of ~3 h due to ubiquitination and lysosomal and proteasomal degradation (Leithe and Rivedal, 2007). Cx43 and Cx46 regulate each other's expression and turnover in a reciprocal manner in addition to their conventional roles as gap junction proteins in lens cells (Banerjee et al., 2011). A mutant form of Connexin 43 causes Oculodentodigital dysplasia (Gabriel et al., 2011). Suppressing the function of Cx43 promotes expression of wound healing-associated genes and hibitits scarring (Tarzemany et al. 2015). Channel conductance and size selectivity are largely determined by pore diameter, whereas charge selectivity results from the amino-terminal domains; transitions between fully open and (multiple) closed states involves global changes in structure of the pore-forming domains (Ek Vitorín et al. 2016). The human Cx43 orthologue is almost identical to the rat protein. It may mediate resistance against the parkinsonian toxin, 1-methyl-4-phenylpyridine (MPP+) which induces apoptosis in neuroblastoma cells by modulating mitochondrial apoptosis (Kim et al. 2016). Dopamine neurons may be the target of MPP+ and play a role in Parkinson's disease. In humans, Cx43 plays roles in the development of the central nervous system and in the progression of glioma (Wang et al. 2017). It interacts with and is regulated by many proteins including NOV (CCN3, IGFBP9; P48745) (Giepmans 2006). Cx43 plays roles in intercellular communication mediated by extracellular vesicles, tunnelling nanotubes and gap junctions (Ribeiro-Rodrigues et al. 2017). Phosphorylation of Cx43 leads to astrocytic coupling and apoptosis, and ultimately, to vascular regeneration in retinal ischemia. Paxillin (Pxn; 591 aas; P49023), a cytoskeletal protein involved in focal adhesion, leads to changes in connexin 43 by direct protein-protein binding, thereby influencing osteocyte gap junction elongation (Zhang et al. 2018). Regulation of Cx43 abundance involves transcriptional/post-transcriptional and translational/post-translational mechanisms that are modulated by an interplay between TGF-beta isoforms and PGE2, IL-1beta, TNF-alpha and IFN-gamma (Cheng et al. 2018). In the developing fetal human kidney, cytoplasmic expression of Cx36 was localized to nephrons in different developmental stages, glomerular vessels and collecting ducts, and of Cx43 was localized to the endothelium of glomerular and peritubular vessels, as well as to the epithelium of the proximal tubules (Ráduly et al. 2019). Mutations in the gap junction protein α1 (GPA1) gene cause oculodentodigital dysplasia (Pace et al. 2019).
Heteromeric connexin (Cx)32/Cx26) (transports cAMP, cGMP and all inositol phosphates with 1-4 esterified phosphate groups (homomeric Cx26(β2) or homomeric Cx32 do not transport the inositol phosphates as well) (Ayad et al., 2006). The GJB2 gene encodes connexin 26, the protein involved in cell-cell attachment in many tissues. GJB2 mutations cause autosomal recessive (DFNB1) and sometimes dominant (DFNA3) non-syndromic sensorineural hearing loss as well as various skin disease phenotypes (Iossa et al., 2011). TMS1 regulates oligomerization and function (Jara et al., 2012). The carboxyl tail pg Cx32 regulates gap junction assembly (Katoch et al. 2015). In Cx46, neutralization of negative charges or addition of positive charge in the Cx26 equivalent region reduced the slow gate voltage dependence. In Cx50 the addition of a glutamate in the same region decreased the voltage dependence and the neutralization of a negative charge increased it. Thus, the charges at the end of TMS1 are part of the slow gate voltage sensor in Cxs. The fact that Cx42, which has no charge in this region, still presents voltage dependent slow gating suggests that charges still unidentified also contribute to the slow gate voltage sensitivity (Pinto et al. 2016). Syndromic deafness mutations at Asn14 alter the open stability of Cx26 hemichannels (Sanchez et al. 2016). The Leu89Pro substitution in the second TMS of CX32 disrupts the trafficking of the protein, inhibiting the assembly of CX32 gap junctions, which in turn may result in peripheral neuropathy (Da et al. 2016). Cx26 mutants that promote cell death or exert transdominant effects on other connexins in keratinocytes lead to skin diseases and hearing loss, whereas mutants having reduced channel function without aberrant effects on coexpressed connexins cause only hearing loss (Press et al. 2017). When challenged by a field of 0.06 V/nm, the Cx26 hemichannel relaxed toward a novel configuration characterized by a widened pore and an increased bending of the second TMS at the level of the conserved Pro87. A point mutation that inhibited such a transition impeded hemichannel opening in electrophysiology and dye uptake experiments. Thus, the Cx26 hemichannel uses a global degree of freedom to transit between different configuration states, which may be shared among all connexins (Zonta et al. 2018). A group of human mutations within the N-terminal (NT) domain of connexin 26 hemichannels produce aberrant channel activity, which gives rise to deafness and skin disorders, including keratitis-ichthyosis-deafness (KID) syndrome. Structural and functional studies indicate that the NT domain of connexin hemichannels is folded into the pore, where it plays important roles in permeability and gating. The mutation, N14K disrupts cytosolic intersubunit interactions and promotes channel opening (Valdez Capuccino et al. 2018). A missense mutation in the Connexin 26 gene is associated with autosomal recessive sensorineural deafness (Leshinsky-Silver et al. 2005).
Connexin45 (cx45; Gap Junction protein γ1; GJγ1; CxG1) of 396 aas and 4 TMSs (Kopanic et al. 2015).
The Mg2+ transporter, MgtE. The crystal structure of the N-terminal hydrophilic domain has been determined to 2.3 Å resolution (Hattori et al., 2007) (>50% identical to 9.A.19.1.1), while the C-terminal transmembrane domain has been determined at 2.2 Å resolution (Takeda et al. 2014). The structure reveals a homoldimer with the channel at the interface of the two subunits. There's a plug at the cytoplasmic face. It can bind Mg2+, Mn2+ and Ca2+.
The sterol (dexamethasone, aldosterone) and low NaCl diet-inducible FXYD domain-containing ion transport regulator 4 precursor (Channel inducing factor, CHIF). It is an IsK-like MinK homologue (Attali et al., 1995). It regulates the Na+,K+-ATPase and the KCNQ1 channel protein as well as other ICNQ channels, opening them at all membrane potentials (Jespersen et al. 2006).
γ-subunit (proteolipid) of Na+,K+-ATPase, FXYD2. Also functions as a cation-selective channel (Sha et al. 2008).
THe Urea transporter channel protein of 337 aas and 11 TMSs. The 3-d structure (2.3 Å resolution) is available (Levin et al., 2009). Urea binding and flux as well as dimethylurea (DMU) transport have been modeled (Zhang et al. 2017).
Proton-gated urea transport channel (UreI) (pH-sensitive). Allows the transmembrane flow of urea, hydroxyurea and (at a low rate) water. KB for urea is ~150mM (Sachs et al., 2006; Scott et al., 2010). Transport kinetics and selectivity have been defined (Gray et al., 2011). The 3-d structure reveals a hexameric protein with a channel included within the twisted 6 TMS bundle of each protomer. It displays a two helix hairpin structure repeated three times around the central axis of the channel (Strugatsky et al. 2012).
Ryanodine receptor Ca2+ release channel, RyR2. Causes Ca2+ release from the E.R. and consequent cardiac arrhythmia (Chelu and Wehrens, 2007). Associates with FKBP12.6, but phosphorylation by protein kinase A on serine-2030 causes dissociation (Jones et al., 2008). An interaction site for FKBP12.6 may be present at the RyR2 C terminus, proximal to the channel pore, a sterically appropriate location that would enable this protein to play a role in the modulation of this channel (Zissimopoulos and Lai 2005).
Enhanced binding of calmodulin corrects arrhythmogenic channel disorder in myocytes (Fukuda et al. 2014). RyR2s can open spontaneously, giving rise to spatially-confined Ca2+ release events known as "sparks." They are organized in a lattice to form clusters in the junctional sarcoplasmic reticulum membrane. The spatial arrangement of RyR2s within clusters strongly influences the frequency of Ca2+ sparks (Walker et al. 2015). Structures of RyR2 from porcine heart in both the open and closed states at near atomic resolutions have been determined using single-particle electron cryomicroscopy (Peng et al. 2016). Structural comparisons revealed breathing motions of the overall cytoplasmic region resulting from the interdomain movements of amino-terminal domains (NTDs), Helical domains, and Handle domains, whereas little intradomain shifts are observed in these armadillo repeat-containing domains. Outward rotations of the central domains, which integrate the conformational changes of the cytoplasmic region, lead to the dilation of the cytoplasmic gate through coupled motions. These observations provide insight into the gating mechanism of RyRs (Peng et al. 2016). RyR2 is subject ot regulation by cytoplasmic Zn2+ (>1nM), and this regulation plays a key role in diastolic SR Ca2+ leakage in cardiac muscle (Reilly-O'Donnell et al. 2017). Ryanodine receptor-mediated SR Ca2+ efflux is apparently balanced by concomitant counterion currents across the SR membrane (Sanchez et al. 2018).
The Ryanodine receptor Ca2+/K+ release tetrameric channel, RyR1, present in skeletal muscle, is 5038 aas long. Mutants are linked to core myopathies such as Central Core Disease, Malignant Hyperthermia and Multiple Minicore Disease) (Xu et al., 2008). RyR1 interacts with CLIC2 to modulate its channel activity (Meng et al., 2009). A model pf RyR1 has been constructed encompassing the six transmembrane helices to calculate the RyR1 pore region conductance, to analyze its structural stability, and to hypothesize the mechanism of the Ile4897 CCD-associated mutation. The calculated conductance of the wild-type RyR1 suggests that the pore structure can sustain ion currents measured in single-channel experiments. Shirvanyants et al. 2014 observed a stable pore structure with multiple cations occupying the selectivity filter and cytosolic vestibule, but not the inner chamber. Stability of the selectivity filter depends on interactions between the I4897 residue and several hydrophobic residues of the neighboring subunit. Loss of these interactions in the case of the polar substitution, I4897T, results in destabilization of the selectivity filter, a possible cause of the CCD-specific reduced Ca2+ conductance. A 4.8 Å structure of the rabbit orthologue in the closed state of this 2.3 MDa tetramer (3757 aas/protomer) reveals the pore, the VIC superfamily fold and a potential mechanism of Ca2+ gating (Zalk et al. 2015). A cryo-electron microscopy analysis revealed the structure at 6.1 Å resolution (Efremov et al. 2015). The transmembrane domain represents a chimaera of voltage-gated sodium and pH-activated ion channels. They identified the calcium-binding EF-hand domain and showed that it functions as a conformational switch, allosterically gating the channel. Malignant hyperthermia-associated RyR1 mutations in the S2-S3 loop confer RyR2-type Ca2+- and Mg2+-dependent channel regulation (Gomez et al. 2016). Structural analyses have elucidated a novel channel-gating mechanism and a novel ion selectivity mechanism for RyR1 (Wei et al. 2016). Samsó 2016 reviewed structural determinations of RyR by cryoEM and analyzed the first near-atomic structures, revealing a complex orchestration of domains controlling channel function. The structural basis for gating and activation have been determined (des Georges et al. 2016). Junctin and triadin bind to different sites on RyR1; triadin plays an important role in ensuring rapid Ca2+ release during excitation-contraction coupling in skeletal muscle. RyR1 structure/functioin has been reviewed (Zalk and Marks 2017). Possibly, luminal Ca2+ activates RyR1 by accessing a cytosolic Ca2+ binding site in the open channel as the Ca2+ ions pass through the pore (Xu et al. 2017).
|1.A.3.2.6||Inositol 1,4,5-trisphosphate receptor type 1 (IP3 receptor isoform 1) (IP3R 1) (InsP3R1) (Type 1 inositol 1,4,5-trisphosphate receptor) (Type 1 InsP3 receptor)||
The flagellar motor (smf-dependent) (PomAB; MotXY) (Okabe et al., 2005). PomB interacts with the third TMS of PomA in the Na+-driven polar flagellum (Yakushi et al. 2004). Sodium-powered stators of the flagellar motor can generate torque in the presence of the sodium channel blocker, phenamil, with mutations near the peptidoglycan-binding region of PomB (Ishida et al. 2019). FliL associates with the flagellar stator in the sodium-driven Vibrio motor (Lin et al. 2018). When the ion channel is closed, PomA and PomB interact strongly. When the ion channel opens, PomA interacts less tightly with PomB. The plug and loop between TMSs 1 and 2 regulate activation of the stator, which depends on the binding of sodium ion to the D24 residue of PomB (Nishikino et al. 2019).
The TonB energy-transducing system. ExbB/D (the putative H+ channel) are listed here; TonB is listed under TC# 2.C.1.1.1. Deletion of the cytoplasmic loop gives rise to immediate growth arrest (Bulathsinghala et al. 2013). The rotational surveillance and energy transfer (ROSET) model of TonB action postulates a mechanism for the transfer of energy from the IM to the OM, triggering iron uptake and concentration in the periplasm (Klebba 2016).
Annexin A1 (McNeil et al., 2006)
Annexin 2 or Annexin A2 (ANXA2) of 339 aas. Forms a tetrameric complex with the S100A10 protein and binds the C-terminus of the AHNAK protein via the N-terminus of annexin 2 (De Seranno et al., 2006). Direct translocation of Annexin 2 to the cell surface occurs by pore-formation. External annexin A2 acts as a plasminogen receptor, able to stimulate fibrinolysis and cell migration (Pompa et al. 2017).
Annexxin of 369 aas. Schistosomiasis, a major parasitic disease of humans, is second only to malaria in its global impact. The disease is caused by digenean trematodes that infest the vasculature of their human hosts. These flukes are limited externally by a body wall composed of a syncytial epithelium, the apical surface membrane, a parasitism-adapted dual membrane complex. Annexins are important for the stability of this apical membrane system. Leow et al. 2013 presented the first structural and immunobiochemical characterization of an annexin from Schistosoma mansoni. The crystal structures of annexin B22 (4MDV and 4MDU) in the apo and Ca2+ bound forms confirmed the presence of the previously predicted α-helical segment in the II/III linker and revealed a covalently linked head-to-head dimer. The dimeric arrangement revealed a non-canonical membrane binding site and a probable binding groove opposite the binding site. Annexin B22 expression correlated with life stages of the parasite that possess the syncytial tegument layer, and ultrastructural localization by immuno-electron microscopy confirmed the occurrence of annexins in the tegument of S. mansoni.
Heat shock protein-70 homologue, DnaK. Although DnaK homologues are ubiquitous, a transport function in eukaryotes, but not in prokaryotes has been demonstrated.
|1.A.33.1.3||Heat shock protein 70(1B)||
The Bacillus SpoIIQ/SpoIIIAH transcompartment channel interconnects the forespore and the mother cell. The activity of sigmaG requires this channel apparatus through which the adjacent mother cell provides substrates that generally support gene expression in the forespore. Flanagan et al. 2016 reported that SpoIIQ is bifunctional, specifically maximizing sigmaG activity as part of a regulatory circuit that prevents sigmaG from activating transcription of the gene encoding its own inhibitor, the anti-sigma factor CsfB.
Divalent metal ion (Mg2+, Ca2+, Ni2+, etc.) transporter of 317 aas and 3 TMSs. The cryo-EM structure shows a pentameric channel with an asymmetric domain structure and featuring differential separations between the trans-segments, probably reflecting mechanical coupling of the cytoplasmic domain to the transmembrane domain and suggesting a gating mechanism (Cleverley et al. 2015).
Magnesium transport protein, CorA. The structure at 2.7 Å resolution is known. The CorA monomer has a C-terminal membrane domain containing two transmembrane segments and a large N-terminal cytoplasmic soluble domain. In the membrane, CorA forms a homopentamer shaped like a funnel which binds fully hydrated Mg2+ in the periplasm (Maguire 2006). A ring of positive charges are external to the ion-conduction pathway at the cytosolic membrane interface, and highly negatively charged helices in the cytosolic domain appear to interact with the ring of positive charge to facilitate Mg2+ entry. Mg2+ ions are present in the cytosolic domain that are well placed to control the interaction of the ring of positive charge and the negatively charged helices, and thus, control Mg2+ entry (Maguire 2006). Gating is achieved by helical rotation upon the binding of a metal ion substrate to the regulatory binding sites. The preference for Co2+ over Mg2+ has been reported to be determined by the presence of threonine side chains in the channel (Nordin et al. 2013), but more recently, Kowatz and Maguire 2018 showed that Co2+ is not a substrate, and that the intersubunit bound Mg2+ is not required for function and does not control the open versus closed states.
Zn2+/Cd2+ efflux system, ZntB. Mg2+ is not transported. Wan et al. 2011 reported crystal structures in dimeric and physiologically relevant homopentameric forms at 2.3 Å and 3.1 Å resolutions, respectively. The funnel-like structure is similar to that of the homologous Thermotoga maritima CorA Mg2+ channel and a Vibrio parahaemolyticus ZntB (VpZntB). However, the central α7 helix forming the inner wall of the StZntB funnel is oriented perpendicular to the membrane instead of the marked angle seen in CorA or VpZntB. Consequently, the StZntB funnel pore is cylindrical, not tapered, which may represent an "open" form of the ZntB soluble domain. There are three Zn2+ binding sites in the full-length ZntB, two of which could be involved in Zn2+ transport.
The ZntB Zn2+/Cd2+ transporter. The 1.9Å structure of the N-terminal cytoplasmic domain of ZntB has been solved (Tan et al., 2009).
Mitochondrial inner membrane Mg2+ channel protein, Mrs2 (Schindl et al., 2007). Mutational analyses have been carried out, suggesting that internal Mg2+ affects intron splicing (Weghuber et al. 2006). MRS2 is involved in mitochondrial Mg2+ homeostasis (Schäffers et al. 2018). The G-M-N motif determines the ion selectivity, likely together with the negatively charged loop at the entrance of the channel, thereby forming the Mrs2p selectivity filter (Sponder et al. 2013).
The CD20 (Cluster of differentiation-20) protein (297 aas and 4 TMSs) is a putative cation channel (B-lymphocyte CD20 antigen) or an indirect regulator of calcium release. The 3-d structure has been determined (Rougé et al. 2020). It is targeted by monoclonal antibodies for the treatment of malignancies and autoimmune disorders. Rituximab (RTX) activates complement to kill B cells. Rougé et al. 2020 obtained a structure of CD20 in complex with RTX, revealing a compact double-barrel dimer bound by two RTX antigen-binding fragments (Fabs), each of which engages a composite epitope. RTX cross-links CD20 into circular assemblies and lead to a structural model for complement recruitment.
Vanilloid receptor subtype 1 (VR1 or TRPV1) (noxious, heat-sensitive [opens with increasing temperatures; e.g., >42°C]; also sensitive to acidic pH and voltage and inflamation; serves as the receptor for the alkaloid irritant, capsaicin, for resiniferatoxin and for endo-cannabinoids (Murillo-Rodriguez et al. 2017). It is regulated by bradykinin and prostaglandin E2) (contains a C-terminal region, adjacent to the channel gate, that determines the coupling of stimulus sensing and channel opening (Garcia-Sanz et al., 2007; Matta and Ahern, 2007). Activated and sensitized by local anesthetics in sensory neurons (Leffler et al., 2008). A bivalent tarantula toxin activates the capsaicin receptor (TRPV1) by targeting the outer pore domain (Bohlen et al., 2010). Single-channel properties of TRPV1 are modulated by phosphorylation (Studer and McNaughton, 2010). TRPV1 mediates an itch associated response (Kim et al., 2011). The thermosensitive TRP channel pore turret is part of the temperature activation apparatus (Yang et al., 2010). Modular thermal sensors in temperature-gated transient receptor potential (TRP) channels have been identified (Yao et al., 2011). TRPV1 opening is associated with major structural rearrangements in the outer pore, including the pore helix and selectivity filter, as well as pronounced dilation of a hydrophobic constriction at the lower gate, suggesting a dual gating mechanism (Cao et al. 2013). Allosteric coupling between upper and lower gates may account for modulation exhibited by TRPV1 and other TRP channels (Liao et al. 2013). Regulates longevity and metabolism by neuropeptides in mice (Riera et al. 2014). The pore of TRPV1 contains the structural elements sufficient for activation by noxious heat (Zhang et al. 2017). In bull sperm, TRPV1 functions in the regulation of motility and the acrosome reaction (Kumar et al. 2019). The dynamics of water in the transmembrane pore of TRPV1 have been studied (Trofimov et al. 2019). TRPV1 - 6 channel subunits do not combine arbitrarily. With the exception of TRPV5 and TRPV6, TRPV channel subunits preferentially assemble into homomeric complexes (Hellwig et al. 2005).
Intestinal endocyte Ca2+ (Sr2+; Ba2+) entry channel, CaT1. Excision of the Trpv6 gene leads to severe defects in epididymal Ca2+ absorption and male fertility as does the single D541A pore mutation (Weissgerber et al., 2012).
The noxious heat (>52°C)-sensitive vanilloid-like receptor cation selective channel, TRPV2. Ca2+-dependent desensitization of TRPV2 channels is mediated by hydrolysis of phosphatidylinositol 4,5-bisphosphate (Mercado et al., 2010). Deleting the first N-terminal 74 residues preceding the ankyrin repeat domain (ARD) shows a key role for this region in targeting the protein to the membrane. Co-translational insertion of the membrane-embedded region occurs with the TM1-TM4 and TM5-TM6 regions assembling as independent folding domains. ARD is not required for TM domain insertion into the membrane (Doñate-Macian et al. 2015). The TRPV2 structure has been solved at 4 Å resolution by cryoEM (Zubcevic et al. 2016). Formation of a physical complex between mouse TRPV2 (GRC) and the mouse RGA protein promotes cell surface expression of TRPV2 (Stokes et al. 2005).
The nociceptive neuron TRPA1 (Trp-ankyrin 1) senses peripheral damage by transmitting pain signals (activated by cold temperatures, pungent compounds and environmental irritants). Noxious compounds also activate through covalent modification of cysteyl residues (Macpherson et al., 2007). TRPA1 is an excitatory, nonselective cation channel implicated in somatosensory function, pain, and neurogenic inflammation. Through covalent modification of cysteine and lysine residues, TRPA1 can be activated by electrophilic compounds, including active ingredients of pungent natural products (e.g., allyl isothiocyanate), environmental irritants (e.g., acrolein), and endogenous ligands (4-hydroxynonenal) (Chen et al., 2008). General anesthetics activate TRPA1 nociceptive ion channels to enhance pain and inflammation (Matta et al., 2008; Leffler et al., 2011). TMS5 is a critical molecular determinant of menthol sensitivity (Xiao et al., 2008) and a variety of inhibitors which are analgesics. Another class of inhibitors are in the thiadiazole structural class of compounds, and they bind to the TRPA1 ankyrin repeat 6 (Tseng et al. 2018). Inhibitors are potential analgesics. The majority of TRPA1 inhibitors interact with the S5 transmembrane helices, forming part of the pore region of the channel. TRPA1 is a component of the nociceptive response to CO2 (Wang et al., 2010). TRPA1 is a polyunsaturated fatty acid sensor in mammals but not in flies and fish (Motter and Ahern, 2012). It is regulated by its N-terminal ankyrin repeat domain (Zayats et al., 2012). Mutations in TrpA1 cause alterred pain perception (Kremeyer et al. 2010). The hop compound, eudesmol, an oxygenated sesquiterpene, activates the channel (Ohara et al. 2015). These channels regulate heat and cold perception, mechanosensitivity, hearing, inflammation, pain, circadian rhythms, chemoreception, and other processes (Laursen et al. 2014). TRPA1 is a polymodal ion channel sensitive to temperature and chemical stimuli, but its resposes are species specific (Laursen et al. 2015). A probable binding site for general anesthetics has been identified (Ton et al. 2017), and specific residues involved in binding of the anesthetic, propofol, are known (Woll et al. 2017). TrpV1 and TrpA1 are inflammatory mediators causing cutaneous chronic itch in several diseases (Xie and Li 2018). TRPA1 is specifically activated by natural products including allyl isothiocyanate (mustard oil), cinnamaldehyde (cinnamon), allicin (garlic) and trans-anethole in Fennel Oil (FO) (Memon et al. 2019). Mutations in TRPA1 result in insensitivity to pain promoting algogens such as capsaicin, acid, and allyl isothiocyanate (AITC), have been documented (Eigenbrod et al. 2019). TRPA1 transduces noxious chemical stimuli into nociceptor electrical excitation and neuropeptide release, leading to pain and neurogenic inflammation. It is regulated by the membrane environment. Startek et al. 2019 found that mouse TRPA1 localizes to cholesterol-rich domains, and that cholesterol depletion decreases channel sensitivity to chemical agonists. Two structural motifs in TMSs 2 and 4 are involved in cholesterol interactions that are necessary for normal agonist sensitivity and plasma membrane localization. TRPA1 is an irritant sensor and a therapeutic target for treating pain, itch, and respiratory diseases. It can be activated by electrophilic compounds such as allyl isothiocyanate (AITC). A class of piperidine carboxamides (PIPCs) are potent noncovalent agonists (Chernov-Rogan et al. 2019). Saikosaponins are channel antogonists (Lee et al. 2019).
The ion channel viral protein U, Vpu of 81 aas and 1 TMS. Vpu(1-32), forms a helix bundle with characteristic open states. Different amilorides inhibit channel activity (Römer et al. 2004). The mutation A18H converts a non-specific channel to a selective proton channel that is sensitive to rimantadine (Sharma et al., 2011). Vpu forms stable pentamers (Padhi et al. 2013). The mechanism of Vpu, a weakly conducting cation-selective channel that assists in detachment of the virion from infected cells, has been proposed (Padhi et al. 2014). Interactions of Vpu with host cellular constituents have been reviewed (González 2015). Vpu forms large homo aggregates of 16 or 32 subunits (Lin et al. 2016).
|1.A.42.1.1||Vpr of HIV||
Fluoride ion channel of 128 aas and 4 TMSs, Fluc or CrcB. The crystal structure is known (PDB5A40; 5A43).
Polycystin 1 (PKD1 or PC1) assembles with TRPP2 (Q86VP3) in a stoichiometry of 3TRPP2: 1PKD1, forming the receptor/ion channel complex (Yu et al., 2009). The C-terminal coiled-coil complex is critical for proper assembly (Zhu et al., 2011). Missense mutations have been identified that affect membrane topogenesis (Nims et al. 2011). Biomarkers for polycystic kidney diseases have been identified (Hogan et al. 2015). Extracellular divalent ions, including Ca2+, inhibit permeation of monovalent ions by directly blocking the TRPP2 channel pore. D643, a negatively charged amino acid in the pore, is crucial for channel permeability (Arif Pavel et al. 2016). Polycystin (TRPP/PKD) complexes, made of transient receptor potential channel polycystin (TRPP)4 and polycystic kidney disease (PKD) proteins, play key roles in coupling extracellular stimuli with intracellular Ca2+ signals. PKD1 and PKD2 form a complex, the structure of which has been solved in 3-dimensions at high resolution. The complex consists of PKD1:PKD2 = 3:1. PKD1 consists of a voltage-gated ion channel fold that interacts with PKD2 to complete a domain-swapped TRP architecture with unique features (Su et al. 2018; Su et al. 2018). The C-terminal tail of PKD1 may play a role in the prognosis of renal disease (Higashihara et al. 2018). TRPP2 uses 2 gating charges found in its fourth TMS (S4) to control its conductive state (Ng et al. 2019). Rosetta structural predictions demonstrated that the S4 undergoes approximately 3- to 5-Å transitional and lateral movements during depolarization coupled to opening of the channel pore. Both gating charges form state-dependent cation-pi interactions within the voltage sensor domain (VSD) during membrane depolarization. The transfer of a single gating charge per channel subunit is required for voltage, temperature, and osmotic swell polymodal gating. Thus, TRPP2 channel opening is dependent on activation of its VSDs (Ng et al. 2019). Polycystin-1 assembles with Kv channels to govern cardiomyocyte repolarization and contractility (Altamirano et al. 2019). Three-dimensional in vitro models answer questions about ADPKD cystogenesis (Dixon and Woodward 2018). The polycystin-1 subunit directly contributes to the channel pore, and its eleven TMSs are sufficient for its channel function (Wang et al. 2019). Polycystin-1 inhibits cell proliferation through phosphatase PP2A/B56alpha (Tang et al. 2019).
Heteromeric polycystic kidney disease proteins 1 and 2-like 1 (PKD1L1/PKD2L1) cation (calcium) channel of kidney primary cilia (DeCaen et al. 2013). PKD2L1 is probably orthologous to mouse TC# 1.A.5.2.2. The voltage dependence of PKD2L1 may reflect the charge state of the S4 domain (Numata et al. 2017). PKD2L1, (TRPP3) is involved in the sour sensation and other pH-dependent processes and is a nonselective cation channel that can be regulated by voltage, protons, and calcium. The 3-d structure has been determined by cryoEM at 3.4 Å resolution (Su et al. 2018). Unlike its ortholog PKD2, the pore helix and TMS6, which are involved in upper and lower-gate opening, adopt an open conformation. The pore domain dilation is coupled to conformational changes of voltage-sensing domains via a series of pi-pi interactions, suggesting a potential PKD2L1 gating mechanism (Su et al. 2018).
Polycystin 2 (PKD2, PC2 or TRPP2) of 968 aas and 8 TMSs (Anyatonwu and Ehrlich, 2005). It is regulated by α-actinin (AAC17470) by direct binding, influencing its channel activity (Li et al., 2007), and is also regulated also by diaphanous-related formin 1 (mDia1) (Bai et al., 2008). It has 8 TMSs with 6 TMSs in the channel domain with N- and C- termini inside (Hoffmeister et al., 2010). PC2 interacts with the inositol 1,4,5-trisphosphate receptor (IP(3)R) to modulate Ca2+ signaling (Li et al. 2009). The PKD2 voltage-sensor domain retains two of four gating charges commonly found in voltage-gated ion channels. The PKD2 ion permeation pathway is constricted at the selectivity filter near the cytoplasmic end of S6, suggesting that two gates regulate ion conduction (Shen et al. 2016). 15% of cases of polycystic kidney disease result from mutations in the gene encoding this protein, while 85% are in PKD1 (Ghata and Cowley 2017). Topological changes between the closed and open sub-conductance states of the functional channel are observed with an inverse correlation between conductance and height of the channel. Intrinsic PC2 mechanosensitivity in response to external forces was also observed (Lal et al. 2018). PC2 is present in ciliary membranes of the kidney and shares a transmembrane fold with other TRP channels as well as an extracellular domain found in TRPP and TRPML channels. Wang et al. 2019 characterized the phosphatidylinositol biphosphate (PIP2) and cholesterol interactions with PC2. PC2 has a PIP binding site close to the equivalent vanilloid/lipid binding site in the TRPV1 channel and a binding site for cholesterol. The two classes of lipid binding sites were compared with sites observed in other TRPs and in Kv channels, suggesting that PC2, in common with other ion channels, may be modulated by both PIPs and cholesterol (Wang et al. 2019).
Phospholamban (PLB or PLN) pentameric Ca2+/K+ channel (Kovacs et al., 1988; Smeazzetto et al. 2013; Smeazzetto et al. 2014). In spite of extensive experimental evidence, suggesting a pore size of 2.2 Å, the conclusion of ion channel activity for phospholamban has been questioned (Maffeo and Aksimentiev 2009). Phosphorylation by protein kinase A and dephosphorylation by protein phosphatase 1 modulate the inhibitory activity of phospholamban (PLN), the endogenous regulator of the sarco(endo)plasmic reticulum calcium Ca2+ ATPase (SERCA). This cyclic mechanism constitutes the driving force for calcium reuptake from the cytoplasm into the myocyte lumen, regulating cardiac contractility. PLN undergoes a conformational transition between a relaxed (R) and tense (T) state, an equilibrium perturbed by the addition of SERCA. Phosphoryl transfer to Ser16 induces a conformational switch to the R state. The binding affinity of PLN to SERCA is not affected ((Kd ~ 60 μM). However, the binding surface and dynamics in domain Ib (residues 22-31) change substantially upon phosphorylation. Since PLN can be singly or doubly phosphorylated at Ser16 and Thr17, these sites may remotely control the conformation of domain Ib (Traaseth et al. 2006). Phospholamban interests with SERCA with conformational memory (Smeazzetto et al. 2017). Under physiological conditions, PLB phosphorylation induces little or no change in the interaction of the TMS with SERCA, so relief of inhibition is predominantly due to the structural shift in the cytoplasmic domain (Martin et al. 2018). The phospholamban pentamer alters the function of the sarcoplasmic reticulum calcium pump, SERCA (Glaves et al. 2019).
Sarcolipin (SLN) anion pore-forming protein of 31 aas and 1 TMS, with selectivity for Cl- and H2PO4-. Oligomeric interactions of sarcolipin and the Ca-ATPase have been documented (Autry et al., 2011). Sarcolipin, but not phospholamban, promotes uncoupling of the SERCA pump (3.A.3.2.7; Sahoo et al. 2013). Forms a pentameric pore that can transport water, Na+, Ca2+ and Cl-. Leu21 serves as the gate (Cao et al. 2015). In the channel, water molecules near the Leu21 pore demonstrated a clear hydrated-dehydrated transition (Cao et al. 2016). Small ankyrin 1 (sAnk1; TC#8.A.28.1.2) and SLN interact with each other in their transmembrane domains to regulate SERCA (TC# 3.A.3.2.7) (Desmond et al. 2017).
The voltage-gated proton channel, mVSOP (269 aas and 2 TMSs) (Sasaki et al., 2006). A hydrophobic plug functions as the gate (Chamberlin et al. 2013). Gating currents reveal that voltage-sensor (VS) activation and proton-selective aqueous conductance opening are thermodynamically distinct steps in the Hv1 activation pathway and show that pH changes directly alter VS activation. Gating cooperativity, pH-dependent modulation, and a high degree of H+ selectivity have been demonstrated (De La Rosa and Ramsey 2018).
The voltage-gated proton channel, Hv1, Hv1 or HVCN1 (273 aas) (Ramsey et al., 2006). Thr29 is a phosphorylation site that activates the HVCN1 channel in leukocytes (Musset et al., 2010). The condctivity pore has been delineated and depends of a carboxyl group (Asp or Glu) in the channel (Morgan et al. 2013). The four transmembrane helices sense voltage and the pH gradient, and conduct protons exclusively. Selectivity is achieved by the unique ability of H3O+ to protonate an Asp-Arg salt bridge. Pathognomonic sensitivity of gating to the pH gradient ensures HV1 channel opening only when acid extrusion will result, which is crucial to its biological functions (DeCoursey 2015). An exception occurs in dinoflagellates (see 1.A.51.1.4) in which H+ influx through HV1 triggers a bioluminescent flash. The gating mechanism of Hv1, cooperativity within dimers and the sensitivity to metal ions have been reviewed (Okamura et al. 2015). How this channel is activated by cytoplasmic [H+] and depolarization of the membrane potential has been proposed by Castillo et al. 2015. The extracellular ends of the first transmembrane segments form the intersubunit interface that mediates coupling between binding sites, while the coiled-coil domain does not directly participate in the process (Hong et al. 2015). Deep water penetration through hHv1 has been observed, suggesting a highly focused electric field, comprising two helical turns along the fourth TMS. This region likely contains the H+ selectivity filter and the conduction pore. A 3D model offers an explicit mechanism for voltage activation based on a one-click sliding helix conformational rearrangement (Li et al. 2015). Trp-207 enables four characteristic properties: slow channel opening, highly temperature-dependent gating kinetics, proton selectivity, and ΔpH-dependent gating (Cherny et al. 2015). The native Hv structure is a homodimer, with the two channel subunits functioning cooperatively (Okuda et al. 2016). Segment S3 plays a role in activating gating (Sakata et al. 2016). Two sites have been identified: one is the binding pocket of 2GBI (accessible to ligands from the intracellular side); the other is located at the exit site of the proton permeation pathway (Gianti et al. 2016). Crystal structures of Hv1 dimeric channels revealed that the primary contacts between the two monomers are in the C-terminal domain (CTD), which forms a coiled-coil structure. Molecular dynamics (MD) simulations of full-length and truncated CTD models revealed a strong contribution of the CTD to the packing of the TMSs (Boonamnaj and Sompornpisut 2018). Histidine-168 is essential for the ΔpH-dependent gating (Cherny et al. 2018). Proton transfer in Hv1 utilizes a water wire, and does not require transient protonation of a conserved aspartate in the S1 transmembrane helix (Bennett and Ramsey 2017). Hv1 channels are present in bull spermatozoa, and these regulate sperm functions like hypermotility, capacitation and acrosome reaction through a complex interplay between different pathways involving cAMP, PKC, and Catsper (Mishra et al. 2019).
The CRAC channel protein, Orai1 (CRACM1) (Prakriya et al. 2006), complexed with the STIM1 or STIM2 protein (Feske et al., 2006). Replacement of the conserved glutamate in the first TMS with glutamine (E106Q) acts as a dominant-negative protein, and substitution with aspartate (E106D) enhances Na+, Ba2+, and Sr2+ permeation relative to Ca2+. Mutating E190Q in TMS3 also affects channel selectivity, suggesting that glutamate residues in both TMS1 and TMS3 face the lumen of the pore (Vig et al. 2006). The Orai1:Stim stoichiometry = 4:2 (Ji et al., 2008). Human Orai1 and Orai3 channels are dimeric in the closed resting state and open states. They are tetrameric when complexed with STIM1 (Demuro et al., 2011). A dimeric form catalyzes nonselective cation conductance in the STIM1-independent mode. STIM1 domains have been characterized (How et al. 2013). Alternative translation initiation of the Orai1 message produces long and short types of Ca2+ channels with distinct signaling and regulatory properties (Desai et al. 2015). STIM2 plays roles similar to STIM1 in regulating basal cytosolic and endoplasmic reticulum Ca2+ concentrations by controling Orai1, 2 and 3. STIM2 may inhibit STIM1-mediated Ca2+ influx. It also regulates protein kinase A-dependent phosphorylation and trafficking of AMPA receptors (TC# 1.A.10) (Garcia-Alvarez et al. 2015). A mechanistic model for ROS (H2O2)-mediated inhibition of Orai1 has been determined (Alansary et al. 2016). Regions that are important for the optimal assembly of hetero-oligomers composed of full-length STIM1 with its minimal STIM1-ORAI activating region, SOAR, have been identified (Ma et al. 2017). Orai1 may be multifunctional (Carrell et al. 2016). Activatioin of Orai1 requires communication between the N-terminus and loop 2 (Fahrner et al. 2017). STIM1 dimers unfold to expose a discrete STIM-Orai activating region (SOAR1) that tethers and activates Orai1 channels within discrete ER-PM junctions (Zhou et al. 2018). SOAR dimer cross-linking leads to substantial Orai1 channel clustering, resulting in increased efficacy and cooperativity of Orai1 channel function. In addition to being an ER Ca2+ sensor, STIM1 functions within the PM to exert control over the operation of SOCs. As a cell surface signaling protein, STIM1 represents a key pharmacological target to control fundamental Ca2+-regulated processes including secretion, contraction, metabolism, cell division, and apoptosis (Spassova et al. 2006). STIM1 also contributes to smooth muscle contractility (Feldman et al. 2017). STIM1-mediated Orai1 channel gating, involves bridges between TMS 1 and the surrounding TMSs 2/3 ring, and these are critical for conveying the gating signal to the pore (Yeung et al. 2018). A review article summarizes the current high resolution structural data on specific EF-hand, sterile alpha motif and coiled-coil interactions which drive STIM function in the activation of Orai1 channels (Novello et al. 2018). Orai1 and STIM1 are involved in tubular aggregate myopathy (Wu et al. 2018). Knowledge of the structure-function relationships of CRAC channels, with a focus on key structural elements that mediate the STIM1 conformational switch and the dynamic coupling between STIM1 and ORAI1 has been discussed (Nguyen et al. 2018). While STIM1 is the native channel opener, a chemical modulator is 2-aminoethoxydiphenyl borate (2-APB) (Ali et al. 2017). ORAI1 channel gating and selectivity iare differentially altered by natural mutations in the first and third transmembrane domains (Bulla et al. 2018). Stim1 responds to both ER Ca2+ depletion and heat, mediates temperature-induced Ca2+ influx in skin keratinocytes via coupling to Orai Ca2+ channels in the plasma membrane, and thereby brings about thermosensing (Liu et al. 2019). Possibly, the interplay between STIM1 alpha3 and Orai1 TM3 allows STIM1 coupling to be transmitted into physiological CRAC channel activation (Butorac et al. 2019).
Ca2+ release-activated Ca2+ (CRAC) channel subunit, Orai, which mediates Ca2+ influx following depletion of intracellular Ca2+ stores. In Greek mythology, the 'Orai' are the keepers of the gates of heaven. The crystal structure (3.35 Å), revealed a hexameric assembly of Orai subunits arranged around a central ion pore which traverses the membrane and extends into the cytosol. A ring of glutamate residues on its extracellular side forms the selectivity filter. A basic region near the intracellular side can bind anions that may stabilize the closed state. The architecture of the channel differs from those of other solved ion channels (Hou et al. 2012). Residues in the third TMS of orai affect the conduction properties of the channel (Alavizargar et al. 2018); a conserved glutamate residue (E262) contributes to selectivity. Mutation of this residue affected the hydration pattern of the pore domain, and impaired selectivity of Ca2+ over Na+. The crevices of water molecules are located to contribute to the dynamics of the hydrophobic gate and the basic gate, suggesting a possible role in channel opening and in selectivity function (Alavizargar et al. 2018).
The Orai channel is characterized by voltage independence, low conductance, and high Ca2+ selectivity and plays a role in Ca2+ influx through the plasma membrane (PM). Liu et al. 2019 reported the crystal structure and cryo-EM reconstruction of a mutant (P288L) channel that is constitutively active. The open state showed a hexameric assembly in which 6 TMS 1 helices in the center form the ion-conducting pore, and 6 TMS 4 helices in the periphery form extended long helices. Orai channel activation requires conformational transduction from TM4 to TM1 and causes the basic section of TM1 to twist outward. The wider pore on the cytosolic side aggregates anions to increase the potential gradient across the membrane and thus facilitate Ca2+ permeation (Liu et al. 2019).
The borine viral diarrhea virus (BVDV) p7 peptide, viral budding process initiator.
Presenilin-1 (PS-1; STM-1; E5-1; AD) Ca2+ leak channel (part of the γ-secretase complex; expression alters the lipid raft composition in neuronal membranes (Eckert and Müller, 2009)). The first 5 TMSs of presenilin-1 are homologous to the 5 TMS CD47 antigenic protein, a constituent of the osteoclast fusion complex (1.N.1.1.1), and CD47 is therefore a presenilin homologue (unpublished observations). The active site of gamma-secretase resides in an aqueous catalytic pore within the lipid bilayer and is tapered around the catalytic aspartates (Sato et al. 2006). TMS 6 and TMS 7 contribute to the hydrophilic pore. Residues at the luminal portion of TMS 6 are predicted to form a subsite for substrate or inhibitor binding on the α-helix facing the hydrophilic milieu, whereas those around the GxGD catalytic motif within TMS 7 are water accessible (Sato et al. 2006).
Presenilin homologue (DUF1119) of 301 aas and 9 TMSs with known 3-d structure. The amino-terminal domain, consisting of TM1-6, forms a horseshoe-shaped structure, surrounding TM7-9 of the carboxy-terminal domain. The two catalytic aspartate residues are located on the cytoplasmic side of TMS 6 and TMS 7, spatially close to each other and approximately 8 Å into the lipid membrane surface. Water molecules gain constant access to the catalytic aspartates through a large cavity between the amino- and carboxy-terminal domains. (Li et al. 2013). Both protease and ion channel activities have been demostrated, and these two activities share the same active site (Kuo et al. 2015). Cleavage is controlled by both positional and chemical factors (Naing et al. 2018).
High affinity copper (Cu+) and silver (Ag+) uptake transporter, Ctr1 of 190 aas and 3 TMSs. The trimeric channel (Eisses and Kaplan, 2005) forms an oligomeric pore with each subunit displaying 3 TMSs and 2 metal binding motifs (Lee et al., 2007). TMS2 is sufficient to form the trimer, and the MXXM motif bind Ag+ (Dong et al. 2015). Ctr1 mediates basolateral uptakes of Cu+ in enterocytes (Zimnicka et al., 2007) and shows copper-dependent internalization and recycling which provides a reversible mechanism for the regulation of cellular copper entry (Molloy and Kaplan, 2009). It acts as a receptor for the two extinct viruses, CERV1 and CERV2 (Soll et al., 2010). Ctr1 takes up platinum anticancer drugs, cisplatin and carboplatin (Du et al., 2012). The 3-d structure is known (Yang et al., 2012). Ctr1 has a low turn over number of about 10 ions/second/trimer (Maryon et al. 2013). Methionine and histidine residues in the transmembrane domain are essential for transport of copper, but when mutated, they stimulated uptake of cisplatin (Larson et al. 2010). Plays important roles in the developing embryo as well as in adult ionic homeostasis (Wee et al. 2013). (-)-Epigallocatechin-3-gallate (EGCG), a major polyphenol from green tea, can enhance CTR1 mRNA and protein expression in ovarian cancer cells. EGCG inhibits the rapid degradation of CTR1 induced by cisplatin (cDDP). The combination of EGCG and cDDP increases the accumulation of cDDP and DNA-Pt adducts, and subsequently enhances the sensitivity of ovarian cancer (Wang et al. 2015). Steroid inhibitors may be able to overcome cycplatin resistance (Kadioglu et al. 2015). ctr1 is upregulated in colorectal cancer cells (Barresi et al. 2016). The N-terminus of CTR1 binds Cu2+ following transfer from blood copper carriers such as human serum albumin to the transporter (Bossak et al. 2018). Once in the cytosol, enzyme-specific chaperones receive copper from the CTR1 C-terminal domain and deliver it to their apoenzymes (Ilyechova et al. 2019). Ctr1 is part of the Sp1-Slc31a1/Ctr1 copper-sensing system, and carnosine, a brain dipeptide, influences copper homeostasis in murine CNS-derived cells (Barca et al. 2019).
The pore-forming peptide, Pep46 (derived from the structural polyprotein (PP) precursor (1012 aas) (Galloux et al. 2007). The 3-D NMR structure of Pep46 is known: Acc# 2IMUA (Galloux et al. 2010).
Epithelial Na+ channel, ENaC (regulates salt and fluid homeostasis and blood pressure; regulated by Nedd4 isoforms and SGK1, 2 and 3 kinases) (Henry et al., 2003; Pao 2012). Cd2+ inhibits α-ENaC by binding to the internal pore where it interacts with residues in TMS2 (Takeda et al., 2007). The channel is regulated by palmitoylation of the beta subunit which modulates gating (Mueller et al. 2010). ENaCs are more selective for Naa+ over other cations than ASICs (Yang and Palmer 2018). ENaC plays a role in chronic obstructive pulmonary diseases (COPD) (Zhao et al. 2014). The hetrodimeric complex can consist of αβγ or δβγ subunits, depending on the tissue (Giraldez et al. 2012). The α- and γ-subunits of the epithelial Na+ channel interact directly with the Na+:Cl- cotransporter, NCC, in the renal distal tubule with functional cosequences, and together they determine bodily salt balance and blood pressure (Mistry et al. 2016). ENaC is regulated by syntaxins (Saxena et al. 2006). The cryoEM structure has been solved (Noreng et al. 2018). Interactions between the epithelial sodium channel gamma-subunit and claudin-8 modulates paracellular sodium permeability in the renal collecting duct (Sassi et al. 2020).
Neuronal acid-sensing cation channel-1, ASIC1 (>90% identical to ASIC1 of Rat (TC#1.A.6.1.2)). 3D structure (1.9Å resolution) has been solved (Jasti et al., 2007). Regulated by the glucocorticoid-induced kinase-1 isoform 1 (SGK1.1) (Arteaga et al., 2008). Residues in the second transmembrane domain of the ASIC1a that contribute to ion selectivity have been defined (Carattino and Della Vecchia, 2012). Outlines of the pore in open and closed conformations describe the gating mechanism (Li et al., 2011). Interactions between two extracellular linker regions control sustained channel opening (Springauf et al., 2011). Can form monomers, trimers and tetramers, but the tetramer may be the predominant species in the plasma membrane (van Bemmelen et al. 2015).
Touch-responsive mechanosensitive degenerin channel complex (Mec-4/Mec-10 form the cation/Ca2+-permeable channel; Mec-2 and Mec-6 regulate) (Bianchi, 2007; Chelur et al., 2002; ). Mec-6 is a chaparone protein required for functional insertion (Matthewman et al. 2018). Mec-10 plays a role in the response to mechanical forces such as laminar shear stress (Shi et al. 2016). MEC-4 or MEC-10 mutants that alter the channel's LSS response are primarily clustered between the degenerin site and the selectivity filter, a region that likely forms the narrowest portion of the channel pore (Shi et al. 2018). TMS2 forms the Ca2+ channel of Mec-4. A C-terminal domain affects trafficking of a neuronally expressed DEG/ENaC. Neuronal swelling occurs prior to commitment to necrotic death (Royal et al. 2005).
Serum paraoxonase/arylesterase 1, PON 1 (Aromatic esterase 1) (A-esterase 1) (Serum aryldialkylphosphatase 1)
|1.A.61.1.1||Chain F or gamma-peptide (44aas; 1TMS), membrane active domain (Bong et al., 1999)||
Bactericidal pore-forming pardaxin (Pa4) permeabilized both lipid and lipopolysaccharide membranes. Five paralogues are known: Pa1, 2, 3, 4, and 5, all nearly identical to each other. The 3-d structure of Pa4 is known. It forms a helix-turn-helix conformation resembling a horseshoe (Bhunia et al., 2010).
ATP-gated P2X3 receptor. Tyr-37 stabilizes desensitized states and restricts calcium permeability (Jindrichova et al., 2011). Exhibits "high affinity desensitization" but slow reactivation from the desensitized state (Giniatullin and Nistri 2013). An endogenous regulator of P2X3 in bladder is the Pirt protein (TC#8.A.64.1.1) Gao et al. 2015). X-ray crystal structures of the human P2X3 receptor in apo/resting, agonist-bound/open-pore, agonist-bound/closed-pore/desensitized and antagonist-bound/closed states have been determined (Mansoor et al. 2016). The open state structure harbours an intracellular motif termed the 'cytoplasmic cap', which stabilizes the open state of the ion channel pore and creates lateral, phospholipid-lined cytoplasmic fenestrations for water and ion egress.
The MerF mercuric ion uptake transporter of 81 aas and 2 TMSs. The NMR structure of the helix-loop-helix core domain of MerF has been determined with a backbone RMSD of 0.58 Å (Howell et al. 2005). Moreover, the fold of this polypeptide demonstrates that the two vicinal pairs of cysteine residues, shown to be involved in the transport of Hg++ across the membrane, are exposed to the cytoplasm. This finding differs from earlier structural and mechanistic models that were based primarily on the somewhat atypical hydropathy plot for MerF and related transport proteins (Howell et al. 2005). The apo state positions one of the cysteine pairs closer to the periplasmic side of the membrane, while in the bound state, the same pair approaches the cytoplasmic side (Hwang et al. 2019). This is consistent with the functional requirement of accepting Hg2+ from the periplasmic space, sequestering it on acceptance, and transferring it to the cytoplasm. Conformational changes in the TMSs facilitate the functional interaction of the two cysteine pairs. Free-energy calculations provide a barrier of 16 kcal/mol for the association of the periplasmic Hg2+-bound protein, MerP, with MerF, and 7 kcal/mol for the subsequent association of MerF's two cysteine pairs (Hwang et al. 2019).
Mg2+ transporter; also called Tumor suppressor candidate 3 isoform a, Tusc3a (69% identity with MagT1) (Zhou and Clapham, 2009).
Glycerol facilitator, GlpF. Transports various polyols with decreasing rates as size increases (Heller et al. 1980); also transports arsenite (As(III) and antimonite (Sb(III)) (Meng et al., 2004). Oligomerization may play a role in determining the rate of transport (Klein et al. 2019).
Aquaporin TIP2-1 (Delta-tonoplast intrinsic protein) (Delta-TIP) (Tonoplast intrinsic protein 2-1) (AtTIP2;1) (Daniels et al. 1996). Transports water and ammonia, and can be activated by mercury (Kirscht et al. 2016). The 3-d structure is known to 1.2Å resolution (Kirscht et al. 2016). It may participate in vacuolar compartmentation and detoxification of ammonium.
Nodulin-26 aquaporin and glycerol facilitator, NIP (de Paula Santos Martins et al. 2015). Transports NH3 5-fold better than water in Hg2+-sensitive fashion (Hwang et al., 2010).
|1.A.8.13.1||MIP family homologue||
Hg2+-inhibitable aquaporin, AqpM (transports both water and glycerol as well as CO2) (Kozono et al., 2003; Araya-Secchi et al., 2011). Its 3-d structure has been determined to 1.7 Å. In AqpM, isoleucine replaces a key histidine residue found in the lumen of water channels, which becomes a glycine residue in aquaglyceroporins. As a result of this and other side-chain substituents in the walls of the channel, the channel is intermediate in size and exhibits differentially tuned electrostatics when compared with the other subfamilies (Lee et al. 2005).
Aquaporin Z water channel (aqpZ gene expression is under sigma S control; induced at the onset of stationary phase) (Mallo and Ashby, 2006). The high resolution 3-d structure is available (PDB 1RC2) revealing two re-entrant coil-helix domains from the selectivity filter (Savage et al. 2003). Coupled mutations enabled glycerol transport (Ping et al. 2018).
Aquaporin, Aqy1 (PIP2-7 7). The subangstron (0.88Å) structure is available (Kosinska Eriksson et al. 2013). the H-bond donor interactions of the NPA motif''s asparagine residues to passing water molecules are revealed. A polarized water-water H-bond configuration is observed within the channel. Four selectivity filter water positions are too closely spaced to be simultaneously occupied. Strongly correlated movements break the connectivity of selectivity filter water molecules to other water molecules within the channel, thereby preventing proton transport via a Grotthuss mechanism.
Aquaporin 1 (CO2-, O2- and nitrous oxide-permeable and water-selective) (Zwiazek et al. 2017). Aquaporin-1 tunes pain perception by interacting with Na(v)1.8 Na+ channels in dorsal root ganglion neurons (Zhang and Verkman, 2010). It is upregulated in skeletal muscle in muscular dystrophy (Au et al. 2008). AQP1 has been reported to first insert as a four-helical intermediate, where helices 2 and 4 are not inserted into the membrane. In a second step this intermediate is folded into a six-helical topology. During this process, the orientation of the third helix is inverted, and it can shift out the membrane core (Virkki et al. 2014). Its synthesis is regluated by Kruppel-like factor 2 (KLF2; Q9Y5W3) which also interacts directly with Aqp1 (Fontijn et al. 2015). A nanoscale ion pump has been derived artificially from Aqp1 (Decker et al. 2017). Mammalian AQP1 channels, activated by cyclic GMP, can carry non-selective monovalent cation currents, selectively blocked by arylsulfonamide compounds AqB007 (IC50 170 muM) and AqB011 (IC50 14 muM). Loop D-domain amino acids activate the channel for ion coductance (Kourghi et al. 2018). Water flux through AQP1s is inhibited by 1 - 10 mμM acetozolaminde (Gao et al. 2006). Aqp1 transports reactive oxygen and nitrogen species (RONS) which may induce oxidative stress in the cell interior. These RONS include both hydrophilic (H2O2 and OH) and hydrophobic (NO2 and NO) RONS (Yusupov et al. 2019). The position of the Arg-195 side chain shows a number of interactions for loop C (Dingwell et al. 2019). AQP1 play vital roles in cellular homeostasis at rest and during endurance running exercises (Rivera and Fahey 2019). AQP1 and AQP4 activities correlate with the severity of hydrocephalus induced by subarachnoid haemorrhage (Long et al. 2019). AQPs are related to osmoregulation and play a critical role in maturation, cryo-stability and motility activation in boar spermatozoa (Delgado-Bermúdez et al. 2019). In foetal kidney, AQP1 expression appeared in the apical and basolateral parts of cells, lining the proximal convoluted tubules and the descending limb of Henle's loop, then in the tubule pole of Bowman's capsule (Ráduly et al. 2019).
The lens fiber MIP aquaporin (Aqp0) of B. taurus (forms membrane junctions in vivo and double layered crystals in vitro that resemble the in vivo junctions). The water pore is closed in the in vitro structure (Gonen et al., 2004b). It interacts directly with the intracellular loop of connexin 45.6 via its C-terminal extension (Yu et al., 2005). Forms human cataract lens membranes (Buzhynskyy et al., 2007; Yang et al., 2011). A mutation that causes congenital dominant lens cataracts has been identified (Varadaraj et al. 2008). AqpO catalyzes Zn2+-modulated water permeability as a cooperative tetramer (Nemeth-Cahalan et al., 2007). It transports ascorbic acid (Nakazawa et al., 2011). The Detergent organization around solubilized aquaporin-0 using Small Angle X-ray Scattering has been reported (Berthaud et al., 2012). Aquaporin 0 (AQP0) in the eye lens is truncated by proteolytic cleavage during lens maturation. This truncated AQP0 is no longer a water channel (Berthaud et al. 2015). A mutation that causes congenital dominant lens cataracts has been identified (Varadaraj et al. 2008). Cataractogenesis in MIP mutants are probably caused by defects in MIP gene expression in mice (Takahashi et al. 2017). An automated data processing and analysis pipeline for transmembrane proteins including Aqp0 in detergent solutions has been presented (Molodenskiy et al. 2020).
Aqp6 aquaporin (also transports NO3- and other anions at acidic pH or in the presence of Hg2+) (Ikeda et al., 2002). AQP6 flicker rapidly between closed and open states. Two well conserved glycine residues: Gly-57 in TMS 2 and Gly-173 in TMS 5 reside at the contact point where the two helices cross. Mammalian orthologs of AQP6 have an asparagine residue (Asn-60) at the position corresponding to Gly-57 in Aqp6. Liu et al. 2005 showed that a single residue substitution (N60G in rat AQP6) eliminates anion permeability but increases water permeability.
Aquaporin-4 (AQP4) is the major water channel in the central nervous system and plays an important role in the brain's water balance, including edema formation and clearance. There are 6 splice variants; the shorter ones assemble into functional, tetrameric square arrays; the longer is palmitoylated on N-terminal cysteyl residues) (Suzuki et al., 2008). The longest, Aqp4e, has a novel N-terminal domain and forms a water channel in the plasma membrane although various shorter variants don't (Moe et al., 2008). AQP4, like AQP0 (1.A.8.8.2), forms water channels but also forms adhesive junctions (Engel et al., 2008) (causes cytotoxic brain swelling in mice (Yang et al., 2008)) Mice lacking Aqp4 have impaired olfactions (Lu et al., 2008). Aqp4 is down regulated in skeletal muscle in muscular dystrophy (Au et al. 2008). The crystal structure is known to 2.8 Å resolution (Tani et al., 2009). The structure reveals 8 water molecules in each of the four channels, supporting a hydrogen-bond isolation mechanism and explains its fast and selective water conduction and proton exclusion (Tani et al., 2009; Cui and Bastien, 2011). It is an important antigen in Neuromyelitis optica (NMO) patients (Kalluri et al., 2011). A connection has been made between AQP4-mediated fluid accumulation and post traumatic syringomyelia (Hemley et al. 2013). AQP4 has increased water permeability at low pH, and His95 is the pH-dependent gate (Kaptan et al. 2015). Also transports NH3 but not NH4+ (Assentoft et al. 2016). Cerebellar damage following status epilepticus involves down regulation of AQP4 expression (Tang et al. 2017). SUR1-TRPM4 and AQP4 form a complex to increase bulk water influx during astrocyte swelling (Stokum et al. 2017). A mutation, S111T, causes intellectual disability, hearing loss, and progressive gait dysfunction (Berland et al. 2018). As in humans, the chicken ortholog, Aqp4, is found in brain > kidney > stomach (Ramírez-Lorca et al. 2006). A Molecular Dynamics Investigation on Human AQP4 has been published (Marracino et al. 2018). AQP1 and AQP4 activities correlate with the severity of hydrocephalus induced by subarachnoid haemorrhage (Long et al. 2019).
Vasopressin-sensitive aquaporin-2 (Aqp2) in the apical membrane of the renal collecting duct (Fenton et al., 2008). Controls cell volume and thereby influences cell proliferation (Di Giusto et al. 2012). It plays a key role in concentrating urine. Water reabsorption is regulated by AQP2 trafficking between intracellular storage vesicles and the apical membrane. This process is tightly controlled by the pituitary hormone arginine vasopressin, and defective trafficking results in nephrogenic diabetes insipidus (NDI). The crystal structure of Aqp2 has been solved to 2.75Å (Frick et al. 2014). In terrestrial vertebrates, AQP2 function is generally regulated by arginine-vasopressin to accomplish key functions in osmoregulation such as the maintenance of body water homeostasis by a cyclic AMP-independent mechanism (Olesen and Fenton 2017; Martos-Sitcha et al. 2015). AQP2 is expressed in the anterior vaginal wall and fibroblasts, and regulates the expression level of collagen I/III i, suggesting that AQP2 is associated with the pathogenesis of stress urinary incontinence through collagen metabolism during ECM remodeling (Zhang et al. 2017). As in humans, the chicken ortholog, Aqp2, is found only in the kidney (Ramírez-Lorca et al. 2006). AQP2 is critical in regulating urine concentrating ability. The expression and function of AQP2 are regulated by a series of transcriptional factors and post-transcriptional phosphorylation, ubiquitination, and glycosylation (He and Yang 2019). Mutation or functional deficiency of AQP2 leads to severe nephrogenic diabetes insipidus, and inhibition of various aquaporins leads to many water-related diseases such as, edema, cardiac arrest, and stroke. Maroli et al. 2019 reported on the molecular mechanisms of mycotoxin (citrinin, ochratoxin-A, and T-2 mycotoxin) inhibition of AQP2 and arginine vasopressin receptor 2 (AVPR2).
Aquaporin 5 (x-ray structure at 2.0 Å resolution (PDB# 3D9S) is available) (Horsefield et al., 2008). Aqp5 is a marker for proliferation and migration of human breast cancer cells (Jung et al., 2011). Plays a role in chronic obstructive pulmonary diseases (COPD) (Zhao et al. 2014). Its expression is regulated by androgens (Pust et al. 2015). As in humans, the chicken ortholog, Aqp5, is found in the intestine, the jejunum, ileum and colon (Ramírez-Lorca et al. 2006). Proteomic analyses of the ocular lens revealed palmitoylation (Wang and Schey 2018). Aquaporin 5 expression correlates with tumor multiplicity and vascular invasion in hepatocellular carcinoma (Vireak et al. 2019).
Polyprotein of 2333 aas (includes the viroporin peptide, NS2B). Viroporin activity for the NS2B protein has been demonstrated (Ao et al. 2015).
Nicotinic acetylcholine-activated cation-selective channel, pentameric α2βγδ (immature muscle) nα2βγδ (mature muscle). A combination of symmetric and asymmetric motions opens the gate, and the asymmetric motion involves tilting of the TM2 helices (Szarecka et al. 2007). Acetylcholine receptor δ subunit mutations underlie a fast-channel myasthenic syndrome and arthrogryposis multiplex congenita (Brownlow et al., 2001; Webster et al., 2012). Residues in TMS2 and the cytoplasmic loop linking TMSs 3 and 4 influence conductance, selectivity, gating and desensitization (Peters et al., 2010). nAChR and TRPC channel proteins (1.A.4) mediate nicotine addiction in many animals from humans to worms (Feng et al., 2006). Cholesterol recognition motifs in transmembrane domains of the human nicotinic acetylcholine receptor have been identified (Baier et al., 2011). Allosteric modulators of the α4β2 subtype of neuronal nicotinic acetylcholine receptors, the dominant type in the brain, are numerous (Pandya and Yakel, 2011). α2β2 and α4β2 nicotinic acetylcholine receptors are inhibited by the β-amyloid(1-42) peptide (Pandya and Yakel, 2011b). The A272E mutation in the alpha7 subunit gives rise to spinosad insensitivity without affecting activation by acetylcholine (Puinean et al. 2012). Inhibited by general anaesthetics (Nury et al., 2011). The X-ray crystal structures of the extracellular domain of the monomeric state of human neuronal alpha9 nicotinic acetylcholine receptor (nAChR) and of its complexes with the antagonists methyllycaconitine and alpha-bungarotoxin have been determined at resolutions of 1.8 A, 1.7 A and 2.7 A, respectively (Zouridakis et al. 2014). Structurally similar allosteric modulators of α7 nAChR exhibit five different pharmacological effects (Gill-Thind et al. 2015). Mutations causing slow-channel myasthenia show that a valine ring in the channel is optimized for stabilizing gating (Shen et al. 2016). Quinoline derivatives act as agonists or antagonists depending on the type and subunit (Manetti et al. 2016). Conformational changes stabilize a twisted extracellular domain to promote transmembrane helix tilting, gate dilation, and the formation of a ""bubble"" that collapses to initiate ion conduction (Gupta et al. 2016). A high-affinity cholesterol-binding domain has been proposed for this and other ligand-gated ion channels (Di Scala et al. 2017). Positive allosteric modulators have been identified (Deba et al. 2018). Menthol stereoisomers exhibit fifferent effects on alpha4beta2 nAChR upregulation and dopamine neuron spontaneous firing (Henderson et al. 2019). Corticosteroids exert direct inhibitory action on the muscle-type AChR (Dworakowska et al. 2018). Both deltaL273F and epsilonL269F mutations impair channel gating by disrupting hydrophobic interactions with neighboring alpha-subunits. Differences in the extent of impairment of channel gating in delta and epsilon mutant receptors suggest unequal contributions of epsilon/alpha and delta/alpha subunit pairs to gating efficiency (Shen et al. 2019). Diffusion dynamics of the gangliosides, GM1s and AChRs is uniformly affected by the intracellular ATP level of a living muscle cell (He et al. 2020).
The α4β2 nicotinic acetylcholine receptor. The NMR structure of the transmembrane domain and the multiple anaesthetic binding sites are known (Bondarenko et al., 2012). Mutations cause autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE; Díaz-Otero et al. 2000).
The alpha7 (α-7) nicotinic acetylcholine receptor (alpha-7 nAcChR) of 502 aas is encoded by the CHRNA7 gene. Acetylcholine binding induces conformational changes that result in open channel formation; opening is blocked by α-bungarotoxin. The protein is a homopentamer. It interacts with RIC3 for proper folding and assembly. The nAChR, but not the glycine receptor, GlyR, exhibits hydrophobic gating (Ivanov et al. 2007). Low resolution NMR structures with associated anesthetics have been reported (Bondarenko et al. 2013). Allosteric modulators exhibit up to 5 distinct pharmacological effects (Gill-Thind et al. 2015). Based on pore hydration and size, a high resolution structure for the channel in the open conformation has been proposed (Chiodo et al. 2015). Agonists reduce dyskinesias in both early- and later-stage Parkinson's disease (Zhang et al. 2015). Monoterpenes inhibit the alpha7 receptor in the order: carveol > thymoquinone > carvacrol > menthone > thymol > limonene > eugenole > pulegone = carvone = vanilin. Among the monoterpenes, carveol showed the highest potency (Lozon et al. 2016). A revised structural model has been proposed (Newcombe et al. 2017). In humans, exons 5-10 in CHRNA7 are duplicated and fused to the FAM7A genetic element, giving rise to the hybrid gene CHRFAM7A. Its product, dupalpha7, is a truncated subunit lacking part of the N-terminal extracellular ligand-binding domain and is associated with neurological disorders, including schizophrenia, and immunomodulation (Lasala et al. 2018). alpha7 and dupalpha7 subunits co-assemble into functional heteromeric receptors, in which at least two alpha7 subunits are required for channel opening. Dupalpha7's presence in the pentameric arrangement does not affect the duration of the potentiated events. Using an alpha7 subunit mutant, activation of (alpha7)2(dupalpha7)3 receptors occurs through ACh binding at the alpha7/alpha7 interfacial binding site (Lasala et al. 2018). B-973 is an efficacious type II positive allosteric modulator (PAM) of alpha7 nicotinic acetylcholine receptors that, like 4BP-TQS and its active isomer GAT107, is able to produce direct allosteric activation in addition to potentiation of orthosteric agonist activity, which identifies it as an ago-PAM (Quadri et al. 2018). DB04763, DB08122 and pefloxacin are antagonists (they are NAMs) while furosemide potentiated ACh responses (it is a Pam) (Smelt et al. 2018). At nM concentration, APPsα (amyloid precursor protein) is an allosteric activator of α7-nAChR, mediated by the C-terminal 16 amino acids (CTα16) (Korte 2019). At µM concentrations, Rice et al. 2019 identified the GABABR1a as a target of APPsα, binding the sushi 1 domain via a 17–amino acid sequence (17-mer). These receptors activate opposing downstream cascades. The intrasubunit cavity of the α7 AcChR is important for the activity of type II positive allosteric modulators while the ECD-TMD junction and intersubunit sites are probably important for the activity of type I positive allosteric modulators (Targowska-Duda et al. 2019). Flavonoids are positive allosteric modulators of alpha7 nicotinic receptors (Nielsen et al. 2019). Active and desensitized state conformations have been examined (Chiodo et al. 2018). Modulators are able to activate or deactivate a7 receptors via allosteric binding; they are called positive allosteric modulators (PAMs) or negative allosteric modulators (NAMs) (Al Rawashdah et al. 2019). Functional divergence related sites cluster in the ligand binding domain, the beta2-beta3 linker close to the N-terminal alpha-helix, the intracellular linkers between transmembrane domains, and the "transition zone" (Pan et al. 2019).
The cation-selective pentameric nicotinic acetylcholine receptor, nAChR, with α (461 aas; P02710), β (493 aas; P02712), γ (506 aas; P02714) and δ (522 aas; P02718) subunits. The transmembrane domain of the uncoupled nAChR adopts a conformation distinct from that of the resting or desensitized state (Sun et al. 2016). Studies with this receptor have been reviewed (Unwin 2013). Many small molecules interact with nAChRs including d-tubocurarine, snake venom protein α-bungarotoxin (α-Bgt), and α-conotoxins, neurotoxic peptides from Conus snails. Various more recently discovered compounds of different structural classes also interact with nAChRs including the low-molecular weight alkaloids, pibocin, varacin and makaluvamines C and G. 6-Bromohypaphorine from the mollusk Hermissenda crassicornis does not bind to Torpedo nAChR but behaves as an agonist on human α7 nAChR (Kudryavtsev et al. 2015). Dimethylaniline mimics the low potency and non-competitive actions of lidocaine on nAChRs, as opposed to the high potency and voltage-dependent block by lidocaine (Alberola-Die et al. 2016). Cholesterol is a potent modulator of the Torpedo nAChR (Baenziger et al. 2017). Cholesterol may play a mechanical role by conferring local rigidity to the membrane so that there is productive coupling between the extracellular and membrane domains, leading to opening of the channel (Unwin 2017). 11beta-(p-azidotetrafluorobenzoyloxy)allopregnanolone (F4N3Bzoxy-AP), a general anesthetic, a photoreactive allopregnanolone analog and a potent GABAAR PAM,was used to characterize steroid binding sites in the Torpedo nAChR in its native membrane environment (Yu et al. 2019). The steroid-binding site in the nAChR ion channel was identified, and additional steroid-binding sites could also be occupied by other lipophilic nAChR antagonists. Structural features of the αM4 TMS determine how lipid dependent changes in alphaM4 structure may ultimately modify nAChR function (Thompson et al. 2020).
Adult strychnine-sensitive glycine-inhibited chloride (anion selective) heteropentameric channel (GlyR; GLRA1) consisting of α1- and β-subunits (Cascio, 2004; Sivilotti, 2010). Ivermectin potentiates glycine-induced channel activation (Wang and Lynch, 2012). Molecular sites for the positive allosteric modulation of glycine receptors by endocannabinoids have been identified (Yévenes and Zeilhofer, 2011). Different subunits contribute asymmetrically to channel conductances via residues in the extracellular domain (Moroni et al., 2011; Xiong et al., 2012). Dominant and recessive mutations in GLRA1 are the major causes of hyperekplexia or startle disease (Gimenez et al., 2012). Open channel 3-d structures are known (Mowrey et al. 2013). Desensitization is regulated by interactions between the second and third transmembrane segments which affect the ion channel lumen near its intracellular end. The GABAAR and GlyR pore blocker, picrotoxin (TC# 8.C.1), prevents desensitization (Gielen et al. 2015). The x-ray structure of the α1 GlyR transmembrane domain has been reported (Moraga-Cid et al. 2015), and residue S296 in hGlyR-alpha1 is involved in potentiation by Delta(9)-tetrahydrocannabinol (THC) (Wells et al. 2015). The structure has also been elucidated by cryo EM (Du et al. 2015) and by x-ray crystalography (Huang et al. 2015). The latter presented a 3.0 A X-ray structure of the human glycine receptor-alpha3 homopentamer in complex with the high affinity, high-specificity antagonist, strychnine. The structure allowed exploration of the molecular recognition of antagonists. Comparisons with previous structures revealed a mechanism for antagonist-induced inactivation of Cys-loop receptors, involving an expansion of the orthosteric binding site in the extracellular domain that is coupled to closure of the ion pore in the transmembrane domain. The GlyR beta8-beta9 loop is an essential regulator of conformational rearrangements during ion channel opening and closing (Schaefer et al. 2017). Association of GlyR with the anchoring protein, gephyrin (Q9NQX3), is due to a hydrophobic interaction formed by Phe 330 of gephyrin and Phe 398 and Ile 400 of the GlyR beta-loop (Kim et al. 2006). Alcohols and volatile anesthetics enhance the function of inhibitory glycine receptors (GlyRs) by binding to a single anaesthetic binding site (Roberts et al. 2006). Aromatic residues in the GlyR M1, M3 and M4 α-helices are essential for receptor function (Tang and Lummis 2018). The neurological disorder, startle disease, is caused by glycinergic dysfunction, mainly due to missense mutations in genes encoding GlyR subunits (GLRA1 and GLRB). Another neurological disease with a phenotype similar to startle disease is a special form of stiff-person syndrome (SPS), which is most probably due to the development of GlyR autoantibodies (Schaefer et al. 2018). GlyRs can be modulated by positive allosteric modulators (PAMs) that target the extracellular, transmembrane and intracellular domains (Lara et al. 2019). Mutations in GLRA1 give rise to hyperekplexia (Milenkovic et al. 2018). Neurosteroid binding sites of GABAARs are conserved in the GlyRs (Alvarez and Pecci 2019). The intracellular domain of homomeric glycine receptors modulates agonist efficacy (Ivica et al. 2020). Inhibitory glycinergic transmission in the adult spinal cord is primarily mediated by glycine receptors (GlyRs) containing the alpha1 subunit. Alpha1ins, a longer alpha1 variant with 8 amino acids inserted into the intracellular large loop between TMSs 3 and 4, is expressed in the dorsal horn of the spinal cord, distributed at inhibitory synapses, and it is engaged in negative control over nociceptive signal transduction. Activation of metabotropic glutamate receptor 5 (mGluR5; TC# 9.A.14.7.1) specifically suppressed alpha1ins-mediated glycinergic transmission and evoked pain sensitization. Extracellular signal-regulated kinase (ERK) was critical for mGluR5 to inhibit alpha1ins. By binding to a D-docking site created by the 8-amino-acid insert ERK catalyzed alpha1ins phosphorylation at Ser380, which favored alpha1ins ubiquitination at Lys379 and led to alpha1ins endocytosis. Disruption of the ERK interaction with alpha1ins blocked Ser380 phosphorylation, potentiated glycinergic synaptic currents, and alleviated inflammatory and neuropathic pain (Zhang et al. 2019). The startle disease mutation (αS270T) affects the opening state for activation of presynaptic homomeric GlyRs, as well as postsynaptic heteromeric GlyRs, but the former are affected more. Both respond to glycine less efficiently (Wu et al. 2020). Cannabinoids exert therapeutic effects on several diseases such as chronic pain and startle disease by targeting glycine receptors (GlyRs). They target a serine residue at position 296 in the third TMS of the alpha1/alpha3 GlyR on the outside of the channel at the lipid interface where cholesterol concentrates. GlyRs are associated with cholesterol/caveolin-rich domains. and cholesterol reduction significantly inhibits cannabinoid potentiation of glycine-activated currents (Yao et al. 2020).
γ-aminobutyric acid (GABA)-inhibited Cl- channel, type A (α-, β- γ-subunit precursors), regulated by GABA receptor accessory protein, GABARAP (Luu et al., 2006). The anti-convulsant stiripentol acts directly on the GABA(A) receptor as a positive allosteric modulator (Fisher 2009). The major central endocannabinoid, 2-arachidonoyl glycerol (2-AG), also directly acts at GABA(A) receptors to potentiate the receptor at low GABA concentrations (Sigel et al., 2011). The recpetor is also allosterically regulated by neurosteroids via TMS1 of the beta subunit (Baker et al. 2010). General anesthetic binding site(s) have been identified (Chiara et al., 2012; Woll et al. 2018). Hydrophobic anions potently and uncompetitively antagonize GABA (A) receptor function (Chisari et al., 2011). Regulated by neurosteroids; activated by pregnenolone and allopregnenalone (Costa et al., 2012). Allopregnanolone and its synthetic analog alphaxalone are GABAAR positive allosteric modulators (Yu et al. 2019). Different subunits contribute asymmetrically to channel conductances via residues in the extracellular domain (Moroni et al., 2011). Potentiated by general anaesthetics (Nury et al., 2011). Both the alpha and beta subunits are important for activation by alcohols and anaesthetics (McCracken et al. 2010). Direct physical coupling between the GABA-A receptor (of 4 TMSs) and the KCC2 chloride transporter underlies ionic plasticity in cerebellar purkinje neurons in response to brain-derived neurotrophic factor (BDNF) (Huang et al. 2013). An anesthetic binding site has been identified (Franks 2015). Desensitization is regulated by interactions between the second and third transmembrane segments which affect the ion channel lumen near its intracellular end. The GABAAR and GlyR pore blocker, picrotoxin (TC# 8.C.1), prevents desensitization (Gielen et al. 2015). The mechanism of action of methaqualone (2-methyl-3-O-tolyl-4(3H)-quinazolinone, Quaalude(R)), a sedative-hypnotic and recreational drug. Methaqualone is a positive allosteric modulator (PAM) at human alpha1,2,3,5beta2,3gamma2S GABAA receptors (GABAARs) expressed, whereas it displays diverse functionalities at the alpha4,6beta1,2,3delta GABAAR subtypes, ranging from inactivity (alpha4beta1delta), through negative (alpha6beta1delta) or positive allosteric modulation (alpha4beta2delta, alpha6beta2,3delta), to superagonism (alpha4beta3delta) (Hammer et al. 2015). The thyroid hormone L-3,5,3'-triiodothyronine (T3) inhibits GABAA receptors at micromolar concentrations and has common features with neurosteroids such as allopregnanolone (ALLOP). Westergard et al. 2015 used functional experiments on alpha2beta1gamma2 GABAA receptors to detect competitive interactions between T3 and an agonist (ivermectin, IVM) with a crystallographically determined binding site at subunit interfaces in the transmembrane domain of a homologous receptor (glutamate-gated chloride channel, GluCl). T3 and ALLOP showed competitive effects, supporting the presence of a T3 and ALLOP binding site at one or more subunit interfaces. Residues in the beta3 subunit, at or near the etomidate/propofol binding site(s), form part of the valerenic acid modulator binding pocket (Luger et al. 2015). IV general anesthetics, including propofol, etomidate, alphaxalone, and barbiturates, enhance GABAA receptor activation. These anesthetics bind in transmembrane pockets between subunits of typical synaptic GABAA receptors (Forman and Miller 2016). Carisoprodol can directly gate and allosterically modulate type A GABA (GABAA) receptors (Kumar et al. 2017). The former sedative-hypnotic and recreational drug methaqualone (Quaalude) is a moderately potent, non-selective positive allosteric modulator of GABAA receptors (GABAARs) (Hammer et al. 2015). A methaqualone analog, 2-phenyl-3-(p-tolyl)quinazolin-4(3H)-one (PPTQ) exhibits intrinsic activity at micromolar concentrations and potentiates the GABA-evoked signaling at concentrations down to the low-nanomolar range (Madjroh et al. 2018). The PPTQ binding site is allosterically linked with sites targeted by neurosteroids and barbiturates. Anesthetic pharmacophore binding has been studied (Fahrenbach and Bertaccini 2018). GABAA receptors are modulated via several sites by GABA, benzodiazepines, ethanol, neurosteroids and anaesthetics among others. Amundarain et al. 2018 presented a model of the alpha1beta2gamma2 subtype GABAA receptor in the APO state and in complex with selected ligands, including agonists, antagonists and allosteric modulators. Sites in TMSs 2 and 3 are important for alcohol-induced conformational changes (Jung and Harris 2006). Many anesthetics and neurosteroids act through binding to the GABAAR transmembrane domainnad x-ray structures have revealed how α-xalone, a neurosteroid anaesthetic, binds and influences potentiation, activation and desensitization (Chen et al. 2018). AA29504 is an allosteric agonist and positive allosteric modulator of GABAA receptors (Olander et al. 2018). Allosteric shift analysis in mutant α1β3γ2L GABAA receptors indicates selectivity and cross-talk among intersubunit transmembrane anesthetic sites (Szabo et al. 2019). Several epilepsy-causing mutations have been identified in the genes of the α1, β3, and γ2 subunits comprising the GABAA receptor (Absalom et al. 2019). Constituents of the GABAA receptor include a transmembrane GARLH/LHFPL protein (TC# 1.A.82.1.7) and the inhibitory synaptic protein, neuroligin 2 (TC# 8.A.117.1.1) (Tomita 2019). GABAA receptors containing mutant alpha5 and alpha1 subunits all had reduced cell surface and total cell expression with altered endoplasmic reticulum processing, impaired synaptic clustering, reduced GABAA receptor function and decreased GABA binding potency. Thus, GABRA5 is a causative gene for early onset epileptic encephalopathy (Hernandez et al. 2019). Mutations at Gln242 or Trp246 that eliminate neurosteroid effects do not eliminate neurosteroid binding within the intersubunit site, but significantly alter the preferred orientation of the neurosteroid (Sugasawa et al. 2019). Binding sites and interactions of propanidid and AZD3043 within GABAAR have been identified (Wang et al. 2018). Clptm1 limits GABAAR forward trafficking from the ER to the plasma membrane, and it regulates inhibitory homeostatic plasticity (Ge et al. 2018). The mechanisms of potentiation and inhibition of GABAA receptors by non-steroidal anti-inflammatory drugs, niflumic and mefenamic acids, have been described (Rossokhin et al. 2019). GABAARs are targets for important classes of clinical agents (e.g., anxiolytics, anticonvulsants, and general anesthetics) that act as positive allosteric modulators (PAMs). PAMs bind selectively to a single intersubunit site in the GABAAR transmembrane domain (Jayakar et al. 2019). The gamma2 subunit is required for clustering of these receptors, for recruitment of the submembrane scaffold protein gephyrin to postsynaptic sites, and for postsynaptic function of GABAergic inhibitory synapses (Alldred et al. 2005). The fourth TMS of the gamma2 subunit is required for postsynaptic clustering, but both the major cytoplasmic loop and the fourth transmembrane domain contribute to efficient recruitment of gephyrin to postsynaptic receptor clusters and are essential for restoration of miniature IPSCs (Alldred et al. 2005). Oligomerization and cell surface expression of recombinant GABAA receptors tagged in the delta subunit have been examined (Oflaz et al. 2019). The isoxazoline ectoparasiticide, fluralaner, exerts antiparasitic effects by inhibiting the function of GABARs, but substitutions of Gly333 in TMS3 led to substantial reductions in the sensitivity to fluralaner (Yamato et al. 2020). A potent photoreactive general anesthetic with novel binding site selectivity for GABAA receptors has been identified (Shalabi et al. 2020). GABAA receptor neurosteroid binding sites have been reviewed (Alvarez et al. 2019). Missense variants in GABRA2 are associated with early infantile epileptic encephalopathy (EIEE) as well as other disorders (Sanchis-Juan et al. 2020).
The GABA receptor consisting of α1, β3, and γ2 subunits. Heteropentameric receptor for GABA, the major inhibitory neurotransmitter in the vertebrate brain. Functions also as the histamine receptor and mediates cellular responses to histamine. Functions as a receptor for diazepines and various anesthetics, such as pentobarbital which bind to separate allosteric effector binding sites. Functions as ligand-gated chloride channel (Jayakar et al. 2015). GABRA1 mutations are associated with familial juvenile myoclonic epilepsy, sporadic childhood absence epilepsy, idiopathic familial generalized epilepsy, infantile spasms and Dravet syndrome. Thus, GABRA1 mutations are associated with infantile epilepsy including early onset epileptic encephalopathies including Ohtahara syndrome and West syndrome (Kodera et al. 2016).
The prokaryotic H+-gated ion channel, GlvI or GLIC (Bocquet et al., 2007), solved at 2.9 Å resolution in the open pentameric state (3EHZ_E) (Bocquet et al., 2009; Corringer et al. 2010). The basis for ion selectivity has been reported (Fritsch et al., 2011). Two stage tilting of the pore lining helices results in channel opening and closing (Zhu and Hummer, 2010). The mechanical work of opening the pore is performed primarily on the M2-M3 loop. Strong interactions of this short and conserved loop with the extracellular domain are therefore crucial to couple ligand binding to channel opening. The H+-activated GLIC has an extracellular domain between TMSs M3 and M4 but lacks the intracellular domain (ICD) which is a distinct folding domain (Goyal et al., 2011). The structural basis for alcohol modulation of GLIC has been reported (Howard et al., 2011). The structure of the M2 TMS indicates that the charge selectivity filter is in the cytoplasmic half of the channel (Parikh et al. 2011). Below pH 5.0, GLIC desensitizes on a time scale of minutes. During activation, the extracellular hydrophobic region undergoes changes involving outward translational movement, away from the pore axis, leading to an increase in pore diameter. The lower end of M2 remains relatively immobile (Velisetty et al., 2012). During desensitization, the intervening polar residues in the middle of M2 move closer to form a solvent-occluded barrier and thereby reveal the location of a distinct desensitization gate. In comparison to the crystal structure of GLIC, the structural dynamics of the channel in a membrane environment suggest a more loosely packed conformation with water-accessible intrasubunit vestibules penetrating from the extracellular end all the way to the middle of M2 in the closed-state (Velisetty et al. 2012). Pore opening and closing is well understood (Zhu and Hummer 2010). X-ray structures of general anaesthetics bound to GLIC reveal a common general-anaesthetic binding site, which pre-exists in the apo-structure in the upper part of the transmembrane domain of each protomer (Nury et al., 2011). Large blockers bind in the center of the membrane, but divalent transition metal ions bind to the narrow intracellular pore entry (Hilf et al., 2010). Alcohols and anaesthetics induce structural changes and activate ligand-gated ion channels of the LIC family by binding in intersubunit cavities (Sauguet et al. 2013; Ghosh et al. 2013). Gating at pH 4 has been visualized by x-ray crystallography (Gonzalez-Gutierrez et al. 2013) Site-directed spin labeling and x-ray analyses have revealed gating transition motions and mechanisms that distinguish active from desensitized states (Dellisanti et al. 2013; Sauguet et al. 2013). Gating involves major rearrangements of the interfacial loops (Velisetty et al. 2014). A single point mutation can change the effect of an anesthetic (desfurane; chloroform) from an inhibitor to a potentiator (Brömstrup et al. 2013). An interhelix hydrogen bond involving His234 is important for stabilization of the open state (Rienzo et al. 2014). The outermost M4 TMS makes distinct contributions to the maturation and gating of the related GLIC and ELIC homologs, suggesting that they exhibit divergent mechanisms of channel function (Hénault et al. 2015). The same allosteric network may underlie the actions of various anesthetics, regardless of binding site (Joseph and Mincer 2016). GLIC and ELIC (TC# 1.A.9.9.1) may represent distinct transmembrane domain archetypes (Therien and Baenziger 2017). Arcario et al. 2017 have demonstrate an anesthetic binding site in GLIC which is accessed through a membrane-embedded tunnel. The anesthetic interacts with a previously known site, resulting in conformational changes that produce a non-conductive state of the channel (Arcario et al. 2017). The gating mechanism has been studied (Lev et al. 2017). R-Ketamine inhibits members of the LIC family, and the structural and dynamics basis for the assymetric inhibitory modulation of ketamine has been revealed (Ion et al. 2017). Residue E35 has been identified as a key proton-sensing residue, as neutralization of its side chain carboxylate stabilizes the active state. Thus, proton activation occurs allosterically at the level of multiple loci with a key contribution of the coupling interface between the extracellular and transmembrane domains (Nemecz et al. 2017). General anesthetics can allosterically favor closed channels by binding in the pore or favor open channels via various subsites in the transmembrane domain (Fourati et al. 2018). GLIC's gating by protonation proceeds by making use of loop F, already known as an allosteric site in other pLGICs, instead of the classic orthosteric site (Hu et al. 2018). Binding of fentanyl to its binding site within GLIC results in conformational changes that inhibit conduction through the channel (Faulkner et al. 2019). This channel and others have been studied by high-speed atomic force microscopy (HS-AFM) which has made it possible to characterized the conformational dynamics of single unlabeled transmembrane channels and transporters (Heath and Scheuring 2019).
The bacterial pentameric Cys-loop ligand-gated ion channel (Erwinia chrysanthemi ligand-gated ion channel), ELIC. A 3.3 Å resolution structure is available (Hilf and Dutzler, 2008; Corringer et al., 2010). X-ray analyses have identified three distinct binding sites for anaesthetics, one in the channel, one at the end of a TMS, and one in a hydrophobic pocket of the extracellular domain (Spurny et al. 2013). Motions involving desensitization have been defined (Dellisanti et al. 2013). Simulations indicate the similarities with and differences between the Acetylcholine receptor (Cheng et al. 2009). This family includes members with very divergent properties (Gonzalez-Gutierrez and Grosman 2015). Cysteamine is an agonist for ELIC (Hénault and Baenziger 2016). X-ray structures and functional measurements support a pore-blocking mechanism for the inhibitory action of short-chain alcohols which bind to the TMSs (Chen et al. 2016). GLIC (TC# 1.A.9.8.1) and ELIC may represent distinct transmembrane domain archetypes (Therien and Baenziger 2017), and both bind hopenoids at the mamalian cholesterol binding site (Barrantes and Fantini 2016). A high-resolution structure of ELIC in a lipid-bound state has revealed a phospholipid binding site at the lower half of pore-forming transmembrane helices M1 and M4 and at a nearby site for neurosteroids, cholesterol or general anesthetics (Hénault et al. 2019). This site is shaped by an M4-helix kink and a Trp-Arg-Pro triad that is highly conserved in eukaryote GABAA/C and glycine receptors. M4 is intrinsically flexible, and M4 deletions or disruptions of the lipid-binding site accelerate desensitization, suggesting that lipid interactions shape the agonist response (Hénault et al. 2019). 1-Palmitoyl-2-oleoyl phosphatidylglycerol (POPG) stabilizes the open state of ELIC relative to the desensitized state by direct binding to specific sites (Tong et al. 2019). The nicotinic acetylcholine receptor from the Torpedo electric organ, when reconstituted in membranes formed by zwitterionic phospholipids alone, exposure to agonist fails to elicit ion-flux activity, and ELIC has a similar lipid sensitivity. Structures of ELIC in palmitoyl-oleoyl-phosphatidylcholine- (POPC-) only nanodiscs in both the unliganded (4.1-Å resolution) and agonist-bound (3.3 Å) states using single-particle cryoEM have been solved (Kumar et al. 2020). The largest differences occur at the level of loop C - at the agonist-binding sites - and the loops at the interface between the extracellular and transmembrane domains (ECD and TMD, respectively). The transmembrane pore is occluded similarly in both structures. POPC-only membranes prevent ECD-TMD coupling so that the "conformational wave" of liganded-receptor gating takes place in the ECD and the interfacial M2-M3 linker, but fails to penetrate the membrane and propagate into the TMD. The higher affinity for agonists, characteristic of the open- and desensitized-channel conformations, results from the tighter confinement of the ligand to its binding site; this limits the ligand's fluctuations, and thus delays its escape into bulk solvent (Kumar et al. 2020).
Non-structural glycoprotein 4, NSP4 or enterotoxin, of 175 aas and 2 TMSs (Hyser et al. 2012). A pentatmeric structure of a 53 aas NSP4 fragment has been solved (3MIW). NSP4 viroporin is involved in activation. It increases the endoplasmic reticulum (ER) permeability, resulting in decreased ER calcium stores and activation of plasma membrane (PM) calcium influx channels, ultimately causing the elevation in cytoplasmic calcium (Hyser et al. 2013). It activates ER calcium store-operated calcium entry (Hyser et al. 2013). NSP4 VPD is a Ca2+/Ba2+-conducting cation-selective viroporin that transports monovalent and divalent cations equally well (Pham et al. 2017).
Agnoprotein viroporin of 71 aas and 1 TMS (Suzuki et al. 2010).
OmpF general porin. OmpF can deliver peptides of >6 KDa (epitopes) including protamine, through the pore lumen from the periplasm to the outside (Housden et al., 2010; Ghale et al. 2014). For cephalosporin antibiotics, the interaction strength series is ceftriaxone > cefpirome > ceftazidime (Lovelle et al. 2011). An unfolded protein such as colicin E9 can thread through OmpF from the outside to reach the periplasm (Housden et al. 2013). Polynucleotides can pass through OmpF (Hadi-Alijanvand and Rouhani 2015). LPS influences the movement of bulk ions (K+ and Cl-), but the ion selectivity of OmpF is mainly affected by bulk ion concentrations (Patel et al. 2016). OMPs such as OmpF cluster into islands that restrict their lateral mobility, while IMPs generally diffuse throughout the cell. Rassam et al. 2018 demonstrated that when transient, energy-dependent transmembrane connections are formed, IMPs become subjugated by the inherent organisation of OMPs, and that such connections impact IMP function. They showed that while establishing a translocon for import, colicin ColE9 sequesters the IMPs of the proton motive force (PMF)-linked Tol-Pal complex into islands mirroring those of colicin-bound OMPs. Through this imposed organisation, the bacteriocin subverts the outer-membrane stabilizing role of Tol-Pal, blocking its recruitment to cell division sites and slowing membrane constriction. The ordering of IMPs by OMPs via an energised inter-membrane bridge represents an emerging functional paradigm in cell envelope biology (Rassam et al. 2018). Colicin E9 (ColE9) disordered regions exploit OmpF for direction-specific binding, which ensures the constrained presentation of an activating signal within the bacterial periplasm (Housden et al. 2018). Anionic lipid binding can prevent closure of OmpF channels, thereby increasing access of antibiotics that use porin-mediated pathways (Liko et al. 2018). OmpF may be the major route of D-lactate/D-3-hydroxybutyrate oligo-ester secretion (Utsunomia et al. 2017). Lipid Headgroup Charge and Acyl Chain Composition Modulate Closure of the channel (Perini et al. 2019). Piperacillin, tazobactam, ampicillin and sulbactam interact strongly with OmpF, and may be transported (Wang et al. 2019). Gating kinetics are governed by lipid characteristics so that each stage of a sequential closure is different from the previous one, probably because of intra- or intermonomeric rearrangements (Perini et al. 2019). OmpF transports fosfomycin (Golla et al. 2019) and bacteriocins into cells. Polypeptide transport/binding processes generate an essentially irreversible, hook-like assembly that constrains an import activating peptide epitope between two subunits of the OmpF trimer (Lee et al. 2020).
PhoE phosphoporin. The 3-d structure is available (PDB#1PHO)
OmpC general porin. Expression of OmpC and OmpF is reciprocally regulated by the EnvZ/OmpR sensor kinase/response regulator system (Egger et al. 1997). Mutants isolated from patients with MDR E. coli, resistant to several antibiotics, showed decreased permeability to these antibiotics (Lou et al. 2011). The diffusion route of the fluoroquinolone, enrofloxacin, through the OmpC porin has been reported (Prajapati et al. 2018).
PorB (Class 2). The 2.3 Å structure has been determined by x-ray crystallography. There are three putative solute translocation pathways through the channel pore: One pathway transports anions nonselectively, one tranports cations nonselectively, and one facilitates the specific uptake of sugars. Regulated by ATP binding (Tanabe et al., 2010). Exhibits voltage-dependent closure (Jadhav et al. 2013).
Anion-selective porin protein 32, Omp32. The structure is known to 1.5 Å resolution (Zachariae et al. 2006).
Nucleoside-specific channel forming protein, Tsx (Benz et al. 1988).
Type π fimbrial usher, PapC. The crystal structure of the PapC usher translocation domain has been solved (Daniels and Normark, 2008; Remaut et al., 2008). The crystal structure of the full-length PapC usher in complex with its cognate PapDG chaperone-subunit complex in a pre-activation state has been solved. This elucidated the molecular details of how the usher is specifically engaged by allosteric interactions with its substrate, preceding activation, and how the usher facilitates the transfer of subunits from the amino-terminal periplasmic domain to the CTDs during pilus assembly (Omattage et al. 2018).
Usher, Caf1A, important for F1 antigen assembly
Fimbial usher protein, FimD
|1.B.12.1.1||Autotransporter of adhesin involved in diffuse adherence, AidA (Charbonneau and Mourez, 2007). Heptosylated on 16 ser and thr residues which is required for adhesion (Charbonneau et al., 2007).||
|1.B.12.1.2||Autoexporter of virulence factor G, VirG or IcsA||
Autoexporter of pertactin, Ptt of 910 aas with a C-terminal β-barrel domain which has been crystalized (Zhu et al. 2007). It is a bacterial adhesin and vaccine target which influences the duration of B. pertussis infections but does not otherwise affect the disease (Vodzak et al. 2016).
|1.B.12.2.3||Autoexporter of Bordetella resistance to killing proteins||
Autoexporter of adhesion and penetration protein
Autoexporter of temperature-sensitive hemagglutinin, a hemoglobin binding protease, Tsh/Hbp (1377 aas) (Jong and Luirink, 2008; Peterson et al., 2006). The pore of the Hbp TD is largely obstructed, but a variant that lacked one amino acid residue from the N-terminus showed the opening and closing of a channel comparable to what was reported for the TD of NalP. Hbp is processed by an autocatalytic intramolecular mechanism resulting in the stable docking of the α-helical plug in the barrel.
Autotransporter of serine protease, EspP (with long N-terminal leader that prevents improper folding in the periplasm) (Szabady et al., 2005; Ieva et al., 2008). Energy for export is provided by the folding of the C-terminal domain (Peterson et al., 2010).
Autotransporter-1, Pet (serine protease; 1295 aas)) (Eslava et al., 1998; Leyton et al., 2010). The first stage of autotransporter folding determines whether subsequent translocation can deliver the N-terminal domain to its functional form on the bacterial cell surface. Paired conserved glycine-aromatic 'mortise and tenon' motifs join neighbouring beta-strands in the C-terminal barrel domain, and mutations within these motifs slow the rate and extent of passenger domain translocation to the surface of bacterial cell (Leyton et al. 2014).
|1.B.12.5.9||Autoexporter of lipase/esterase, EstA||
Autoexporter of vacuolating cytotoxin, VacA or Vac2, of 1287 aas.
Autotransporter-1, TibA (989 aas; an Adhesin/Invasin associated with some enterotoxigenic E. coli) (Lindenthal and Elsinghorst et al., 1999; Klemm et al. 2006).
Autotransporter of N-terminal protease passenger domain that cleaves surface-localized virulence factors. The 3-d structure is known (Oomen et al., 2004). The crystal structure of the NalP translocator domain revealed a 12 β-stranded transmembrane beta-barrel containing a central alpha-helix. The transmembrane beta-barrel is stable even in the absence of the alpha-helix. Removal of the helix results in an influx of water into the pore region, suggesting the helix acts as a 'plug' (Khalid and Sansom 2006). The dimensions of the pore fluctuate, but the NalP monomer is sufficient for the transport of the passenger domain in an unfolded or extended conformation (Khalid and Sansom 2006). NalP is subject to phase variation (Oldfield et al. 2013).
Alginate export porin, AEP or AlgE (Rehm et al. 1994). A monomeric 18 stranded beta-barrel that is part of a multicomponent, two membrane, envelope-spanning complex that includes AlgK, AlgX and Alg44 (Rehman and Rehm 2013).
|1.B.14.1.14||Ferric-pseudobactin 358 receptor||
FhuA ferrichrome (also albomycin and rifamycin; Colicin M; Microcin J25; Phage T5) receptor (transports phage T1, T5 and φ80 DNA across the outer membrane, dependent on DcrA (SdaC; TC #2.A.42.2.1) and DcrB) (Forms a complex with and acts with TonB and FhuD (the periplasmic binding receptor (3.A.1.14.3) to deliver siderophore to FhuD (Carter et al., 2006; Braun et al., 2009)). Deletion of the 160-residue cork domain and five large extracellular loops converted this non-conductive, monomeric, 22-stranded beta-barrel protein into a large-conductance protein pore (Wolfe et al. 2015).
The iron-citrate receptor/transporter, FecA. TonB mediates both signaling and transport by unfolding portions of the transporter (Mokdad et al. 2012). The ferric citrate regulator, FecR, is translocated across the bacterial inner membrane via a unique Twin-arginine transport dependent mechanism (Passmore et al. 2020).
FepA ferri-enterobactin (also Colicins B and D) receptor for the 37 aas disulfide-containing K+ channel toxin, BgK (Braud et al., 2004). Functions by a "ball and chain" mechanism; The transport process involves expulsion of the N-terminal globular domain from the C-terminal beta-barrel (Ma et al. 2007). Conformational rearrangements occur in the N-terminus of FepA during FeEnt transport, but disengagement of the N-domain, out of the rigid channel suggests that it remains within the transmembrane pore as FeEnt enters the periplasm (Majumdar et al. 2020).
CirA Fe3+-catecholate receptor. Serves as the receptor for the TonB- and proton-dependent uptake of the E. coli bacteriocin, Microcin L (MccL) (Morin et al., 2011). CirA is also the translocator for colicin Ia (Jakes and Finkelstein, 2010). Plays roles in cefiderocol and ceftazidime resistance (Ito et al. 2018).
Ferripyoverdine/pyocin S3 receptor, FpvA (Adams et al., 2006; Nader et al., 2007; Schalk et al., 2009; Nader et al., 2011)
The Ferripyochelin receptor, FptA (Michel et al., 2007). In addition to Fe3+, FptA takes up Co2+, Ga3+, and Ni2+ at low rates (Braud et al., 2009). The high resolution 3-d structure of FptA (2.0 Å) bound to iron-pyochelin has been solved (Cobessi et al. 2005). The pyochelin molecule provides atetra-dentate coordination of iron. The structure is typical of the TonB-dependent receptor/transporter superfamily.
Heme/hemoglobin receptor of 660 aas and 22 C-terminal β-strands with an N-terminal "plug" domain, ShuA. The 3-d structure is known to 2.6 Å resolution, revealing the histidyl residues in the barrel and plug that can interact with heme (Cobessi et al. 2010).
|1.B.14.2.9||Probable TonB-dependent receptor NMB0964||
BtuB cobalamin receptor (also transports phage C1 DNA across the outer membrane). Two Ca2+ binding sites in BtuB mediate cobalamine binding (Cadieux et al., 2007). Cobalamine uptake into the periplasm is reversible, but efflux is pmf-independent (Cadieux et al., 2007). The 3-d structure is available (PDB#1NQE). The Ton box and the extracellular substrate binding site are allosterically coupled (bidirectional), and TonB binding may initiate a partial round of transport (Sikora et al. 2016). Substrate binding to the extracellular surface of the protein triggers the unfolding of an energy coupling motif at the periplasmic surface. Thus, substrate binding reduces the interaction free energy between certain residues, thereby triggering the unfolding of the energy coupling motif (Lukasik et al. 2007). Multiple extracellular loops contribute to substrate binding and transport by BtuB (Fuller-Schaefer and Kadner 2005).
HasR receptor-HasA haemophore heme receptor complex (HasA, an extracellular heme binding protein, binds one heme and transfers it directly to HasR, which uses HasB (2.C.1.1.2) (a TonB homologue) instead of TonB (2.C.1.1.1) for energization) (Benevides-Matos et al., 2008; Izadi-Pruneyre et al., 2006; Lefèvre et al., 2008; Benevides-Matos and Biville, 2010). A signaling domain in HasR interacts with a partially unfolded periplasmic domain of an antisigma factor, HasS, to control transcription by an ECF sigma factor (Malki et al. 2014). The HasR domain responsible for signal transfer is highly flexible in two stages of signaling, extends into the periplasm at about 70 to 90 A from the HasR beta-barrel and exhibits local conformational changes in response to the arrival of signaling activators (Wojtowicz et al. 2016).
FyuA Fe3+-yersiniabactin and pesticin (Psn; a bacteriocin) receptor and uptake protein of 673 aas. It contributes to biofilm formation and infection (Hancock et al., 2008).
TolC outer membrane exporter of hemolysin, drugs, siderophores such as enterobactin, etc. (Bleuel et al., 2005). The 3-d structure is available (PDB#1EK9). The three monomers form a continuous channel, and each monomer contributes 4 β-strands to the 12 stranded β-barrel (Koronakis et al. 2000). The Salmonella enterica subspecies Typhi homologue is the ST50 antigen (G4C2H4) used in tests for typhoid fever, and a 2.98 Å resolution structure revealed a trimer that forms an alpha-helical tunnel and a beta-barrel transmembrane channel traversing the periplasmic space and outer membrane, respectively (Guan et al. 2015). K. pneumoniae TolC plays a role in resistance towards most antibiotics, suggesting that it can interact with the AcrB efflux pump (Iyer et al. 2019).
CusC outer membrane exporter of copper ion, Cu+, and silver ion Ag+. The crystal structure of CusC is known (Lei et al. 2013) providing evidnce concerning the folding mechanism giving rise to the channel.
Outer membrane hemagglutinin secretion protein, FhaC. Functionally important conserved motifs have been identified (Delattre et al., 2010). The x-ray structure reveals a beta-barrel pore obstructed by two structural elements conserved in all two partner secretion systems, an N-terminal α-helix and an extracellular loop. FhaC goes from the closed to the open state in the presence of the filamentous haemagglutinin adhesin, FHA. The N-terminal α-helix is displaced into the periplasm during FHA secretion (Guérin et al. 2014). With two POTRA domains in the periplasm, a transmembrane beta barrel and a large loop harboring a functionally important motif, FhaC epitomizes the conserved features of the superfamily (Jacob-Dubuisson et al. 2009). The conserved secretion domain of FHA interacts with the POTRA domains, specific extracellular loops and strands of FhaC and the inner beta-barrel surface. The interaction map indicates a funnel-like pathway, with conformationally flexible FHA entering the channel in a non-exclusive manner and exiting along a four-stranded beta-sheet at the surface of the FhaC barrel. This sheet of FhaC guides the secretion domain of FHA along discrete steps of translocation and folding (Baud et al. 2014). The membrane-proximal POTRA domain exists in several conformations, and the binding of FHA displaces this equilibrium (Guérin et al. 2015).
Non-specific, 14 β-stranded monomeric OmpG porin (Conlan et al. 2000). pH-induced conformational changes of OmpG have been studied after reconstitution in native E. coli lipids (Mari et al., 2010). Encoded by a gene in a gene cluster also encoding an ABC sugar uptake system (TC# 3.A.1.1.46), a glucosyl hydrolase and two oxidoreductases. Therefore it's phsiological function may be glucoside uptake. At neutral/high pH, the channel is open and permeable to substrates of size up to 900Da. At acidic pH, loop L6 folds across the channel and blocks the pore. The channel blockage at acidic pH appears to be triggered by the protonation of a histidine pair on neighboring β-strands, which repel one another, resulting in the rearrangement of loop L6 and channel closure (Köster et al. 2015). Crystallization and analysis by electron microscopy and X-ray crystallography revealed the fundamental mechanisms essential for channel activity. A 28 aa extension has been added to the 14 β-TMS barrel to make a 16 β-TMS barrel with normal activity and stability but differing pH sensitivity (Korkmaz et al. 2015). A minimized OmpG porin of only 220 aas still exhibits gating, but it was 5-fold less frequent than in native OmpG. The residual gating of the minimal pore is independent of L6 rearrangements and involves narrowing of the ion conductance pathway, most probably driven by global stretching-flexing deformations of the membrane-embedded β-barrel (Grosse et al. 2014). pH-dependent gating is controlled by an electrostatic interaction network formed between the gating loop and charged residues in the lumen (Perez-Rathke et al. 2018). 3-d structures of the protein in lipid bilayers have been solved (Retel et al. 2017). OmpG may provide a route for D-lactate/D-3-hydroxybutyrate oligo-ester secretion as well as sugar uptake (Utsunomia et al. 2017). OmpG lacks a central constriction and has an exceptionally wide pore diameter of about 13 Å. The equatorial plane of OmpG harbors an annulus of four alternating basic and acidic patches, and manipulation of charge distribution in the arginine and glutamate clusters alters sugar specificity and ion selectivity (Schmitt et al. 2019).
|1.B.22.1.2||XcpQ secretin protein||
|1.B.22.3.2||InvG invasion protein secretin||
M. smegmatis porin, MspA (cation selective due to a high density of negative charges in the constriction zone, but it transports glucose, serine, hydrophilic β-lactams and (slowly) phosphate (Wolschendorf et al., 2007)) The MspC paralogue appears to have the same specificity as MspA. Both can also transport fluoroquinolones and chloramphenicol but not the larger erythromycin, kanamycin, and vancomycin (Danilchanka et al., 2008). Also allows uptake of ferric iron (Jones and Niederweis, 2010). The 3-d structure is known (PDB#1UUN). It is a β-barrel with N- and C-termini of their single hairpins on the outside, and their chains run in an anti-clockwise direction around the central pore. Both of these characteristics are opposite in most gram-negative bacterial β-barrels (Remmert et al., 2010). Forms octameric voltage-gated nanopores where each subunit contributes 2 TMSs to the 16 stranded β-barrel (Faller et al. 2004; Rodrigues et al. 2011; Pavlenok et al. 2012) that can be used for nanopore sequencing (Laszlo et al. 2016).
OprD2; OccD1; porin D transports cationic amino acids, peptides and other compounds: lysine, arginine, histidine, ornithine, basic di- and tri-peptides, and cationic antibiotics such as imipenem (n-formimidoylthienamycin) and other penems and carbapenems (Tamber et al., 2006). The 3-d structure and drugs transported are known (4FOZ; Parkin and Khalid 2014). OprD is the vitronectin receptor. Vitronectin enhances P. aeruginosa adhesion to host epithelial cells and thereby enhances virulence (Paulsson et al. 2015). Loss promotes carbapenem resistance (Shen and Fang 2015; Cavalcanti et al. 2015). Loss results in resistance to meropenem (Fluit et al. 2019).
A tricarboxylate transporting porin, OdpH (Occk5) induced by and transports cis-aconitate, isocitrate and citrate; exhibits a large single channel conductance (Tamber et al., 2006; 2007). This porin exhibits a high degree of anion selectivity, and the outer core and O-antigens of LPS sterically occlude the channel entrance to decrease the diffusion constants of approaching ions (Lee et al. 2018).
OpdC or OccD2 histidine-selective porin (Tamber et al., 2006). The 3-D structure and substrate spcificities are known (PDB 3SY9; Eren et al. 2012).
OdpF (OccK2) glucuronate-selective porin; may also transport benzoate and vanillate (Eren et al., 2012). 3-d structure is known (3SZD).
|1.B.25.1.7||OpdO pyroglutamate-specific porin (Tamber et al., 2006)||
Anion-selective OpdK (OccK1 or OpdK) benzoate/vanillate-selective porin (Tamber et al., 2006; Eren et al., 2012; Liu et al. 2012). The structure of the OpdK porin, specific for vanillate and related small aromatic acids, has been solved by x-ray crystallography (3SYS_A). It is a labile trimer with monomers of an 18 β-stranded barrel and with an inner diameter of 8Å (Biswas et al., 2008). Other substrates transported (but less well) include 4-nitrobenzoate, caproate, octanoate, carbenicillin, cefoxitin, tetracycline antibiotics, and carbapenem antibioitics (imipenem and meropenem) (Eren et al., 2012). Molecular dynamic simulations and mutant analyses have been reported (Wang et al. 2012).
|1.B.25.1.9||OpdP glycine-glutamate-selective porin (Tamber et al., 2006)||
Cyclodextrin (high affinity)/linear malto-oligosaccharide (low affinity) porin, CymA (Orlik et al. 2003). Electroosmosis influences the transport efficiency of cyclodextrins through the CymA pore (Bhamidimarri et al. 2016).
LamB (MalL) maltoporin (maltose–maltoheptose). Also catalyzes the uptake of antibiotics (Lin et al. 2014). LamB preferentially binds maltodextrins from the periplasmic side, and thus, sugar binding and uptake are asymmetric (Mulvihill et al. 2019).
Oligosaccharide porin, ScrY (transports sucrose, raffinose and maltooligo-saccharides) (Kim et al. 2002). The 3-d structure known (PDB ID 1A0S). Sucrose translocation through the pore showed two main energy barriers within the constriction region of ScrY. Three asparate residues are key residues, opposing the passage of sucrose, all located within the L3 loop (Sun et al. 2016).
Omp85 outer membrane OMP translocase, YaeT. The high resolution 3-d structure of the N. gonorrhoea orthologue has been solved (Noinaj et al. 2013).
Outer membrane biogenesis complex (Wu et al., 2005). YaeT (BamA) may serve as an outer membrane ""receptor"" for the CdiA/CdiB 2-partner secretion system that mediates direct cell-cell contact-dependent growth inhibition (Aoki et al., 2008). High-resolution structures of crystal forms of BamA POTRA4-5 from E. coli has been reported (Zhang et al., 2011; Sinnige et al. 2014). Solid-state NMR on BamA, a large multidomain integral membrane protein, revealed dynamic conformational states (Renault et al., 2011). In contrast to the N-terminal periplasmic polypeptide-transport-associated (POTRA) domains, the C-terminal transmembrane β-barrel domain of BamA is mechanically much more stable. Exposed to mechanical stress, this β-barrel stepwise unfolds β-hairpins until unfolding has been completed. The mechanical stabilities of β-barrel and β-hairpins are thereby modulated by the POTRA domains, the membrane composition and the extracellular lid closing the β-barrel. The NMR structure of SmpA (OmlA) is also known (Vanini et al. 2006). The periplasmic region of BamA is firmly attached to the β-barrel and does not experience fast global motion around the angle between POTRA 2 and 3, but the barrel is flexible (Sinnige et al. 2014). It appears that the BAM complex does not catalyze insertion and assembly of all out membrane (α- and β-)porins (Dunstan et al. 2015). YfgL shows significant sequence similarity (e-9) with YxaL/K of Bacillus subtilis. The E. coli periplasmic chaperones, Skp and SurA, and BamA, the central subunit of the BAM complex, have been examined with respect to the folding kinetics of a model OMP (tOmpA) (Schiffrin et al. 2017), showing that prefolded BamA promotes the release of tOmpA from Skp, despite the nM affinity of the Skp for tOmpA. This activity is located in the BamA β-barrel domain, but is greater when full-length BamA is present, indicating that both the beta-barrel and POTRA domains are required for maximal activity. By contrast, SurA is unable to release tOmpA from Skp, providing direct evidence against a sequential chaperone model. BamA has a greater catalytic effect on tOmpA folding in thicker bilayers, suggesting that BAM catalysis involves lowering the kinetic barrier imposed by the hydrophobic thickness of the membrane (Schiffrin et al. 2017). While BamA is the primary translocator, TamB is involved in folding and maturation of autotransporters (Babu et al. 2018).
TamA (YftM) of 577 aas; has a 16 transmembrane β-stranded β-barrel with 3 PORTRA domains. The 2.3 Å crystal structure is known revealing that the barrel is closed by a lid-loop (Gruss et al. 2013). The C-terminal β-strand of the barrel forms an unusual inward kink, which weakens the lateral barrel wall and creates a gate for substrate access to the lipid bilayer. TamA is an Omp85 homologue that may function in autotransporter biogenesis together with TamB (TC# 1.B.22.1.2) and OMP85 (Selkrig et al. 2012). The TAM complex likely evolved from an original combination of BamA and TamB, with a later gene duplication event of BamA, giving rise to an additional Omp85 sequence that evolved to be TamA in Proteobacteria and TamL in Bacteroidetes/Chlorobi (Heinz et al. 2015). Possibly TamB nucleates folding of the passenger domain while TamA/B-BamA interact to catalyze β-domain membrane insertion and pore enlargement to facmilitate translocation of partially folded autotransporters (M. Babu et al., unpublished hypothesis).
The oligogalacturonate-specific porin, KdgM. The 3-D structure is known at 1.9 Å resolution (Hutter et al. 2014). KdgM folds into a 12-stranded antiparallel beta-barrel with a circular cross-section defining a transmembrane pore with a minimal radius of 3.1 Å. Most loops that face the cell exterior in vivo are disordered but nevertheless mediate contact between densely packed membrane-like layers in the crystal. The channel is lined by two tracks of arginine residues facing each other across the pore, a feature that is conserved within the KdgM family and is likely to facilitate the diffusion of acidic oligosaccharides (Hutter et al. 2014).
The N-acetylneuraminic acid-inducible, anion selective porin, NanC (Condemine et al., 2005). A crystal structure (3.3 Å resolution) is available (2WJQ; Wirth et al., 2009). It forms a 28 Å high 12 stranded β barrel like the autotransporter, NalP. The pore is lined by basic residues (conserved in other KdgM family members) allowing diffusion of acidic oligosaccharides (Wirth et al., 2009). Single channels of NanC at pH 7.0 have: (1) conductance 100 to 800 pS in 100 mM: KCl to 3 M: KCl), (2) anion over cation selectivity, and (3) two forms of voltage-dependent gating (channel closures above 200 mV). Phosphate interferes with channel conductance (Giri et al. 2012).
The anaerobically induced outer membrance porin, OprG. Transports small neutral amino acids (Kucharska et al. 2015). The 3-d structure is available (Touw et al. 2010). Essential for normal biofilm formation (Ritter et al. 2012). It is an eight-stranded β-barrel monomer that is too narrow to accommodate even the smallest transported amino acid, glycine, raising the question of how OprG facilitates amino acid uptake (Sanganna Gari et al. 2018). Pro-92 of OprG is important for amino acid transport, with a P92A substitution inhibiting transport and the NMR structure of this variant revealing that this substitution produces structural changes in the barrel rim and restricts loop motions. OprG assembles into oligomers in the OM whose subunit interfaces could form a transport channel, and conformational changes in the barrel-loop region may be crucial for its activity (Sanganna Gari et al. 2018).
Outer membrane porin, OmpW, of 212 aas. Mediates transport of quaternary cationic ammonium compounds (Beketskaia et al. 2014). It is involved in anaerobic carbon and energy metabolism, mediating the transition from aerobic to anaerobic lifestyles (Xiao et al. 2016).
|1.B.40.1.1||YadA consists of 3 domains: an adhesion head, a stalk involved in serum resistance, and an anchor that forms a pore for auto-transport (Grosskinsky et al., 2007).||
|1.B.41.1.1||Outer mycolate membrane porin, PorB||
LPS export porin complex, LptBCFG-A-DE, consists of LptD (Omp; OmpA; 784 aas)-LptE (RlpB; 193 aas; O.M. lipoprotein)-LptA (KdsD; YhbN; OstA small; 185 aas periplasmic chaparone protein)-LptB (KdsC; YhbG; 241 aas cytoplasmic ABC-type ATPase)-LptC (YrbK, 199aas;1 N-terminal TMS)- LptFG, part of the ABC transporter. LptDE (1:1 stoichiometry) comprise a two-protein β-barrel-lipoprotein complex in the outer membrane that assembles and exports LPS (Chng et al., 2010). After LPS (or a precursor) is transported across the inner membrane by MsbA (3.A.1.106.1), this seven component system translocates LPS from the outer surface of the inner membrane to the outer surface of the outer membrane using ATP hydrolysis to sequentially energize transfer from one binding site to another in several steps (Freinkman et al. 2012; Okuda et al. 2012; Sherman et al. 2014). LPS interacts with LptC and LptA sequentially before being passed to the LptD outer membrane porin, anchored by the LptE lipoprotein on the inner surface of the outer membrane. LptF and LptG are the transmembrane consituents of the ABC pump, and LptB is the ATPase of an ABC-like system that energizes the transport using several ATP molecules (Okuda et al. 2012; Sherman et al. 2014). LptC interconnects the LptBFG ABC system with the periplasmic LptA protein via its large periplasmic domain (Villa et al. 2013). LptDE form a complex in the outer membrane which inserts LPS into this membrane. The 3-D strcture of the complex shows that the LptE lipoprotein inserts into the 26 stranded barrel of LptD as a plug. The first two strands of LptD contain prolines and are therefore distorted, possibly creating a portal for lateral diffusion of LPS into the outer leaflet of the outer membrane (Qiao et al. 2014). The 3-d structure of the Pseudomonas aeruginosa LptA, LptH, has been solved at 2:75 Å resolution revealing a β-jellyroll fold similar to that in LptD (Bollati et al. 2015). Direct interaction of LptB and LptC has been demonstrated (Martorana et al. 2016). A specific binding site in the LptB ATPase for the coupling helices of the transmembrane LptFG complex is responsible for coupling ATP hydrolysis by LptB with LptFG function to achieve LPS extraction (Simpson et al. 2016). After biosynthesis, bacterial lipopolysaccharides (LPS) are transiently anchored to the outer leaflet of the inner membrane (IM). The ABC transporter LptB2FG extracts LPSs from the IM and transports them to the outer membrane. Luo et al. 2017 reported the crystal structure of nucleotide-free LptB2FG from P. aeruginosa. It shows that LPS transport proteins LptF and LptG each contain a TM domain (TMD), a periplasmic beta-jellyroll-like domain and a coupling helix that interacts with LptB on the cytoplasmic side. The LptF and LptG TMDs form a large outward-facing V-shaped cavity in the IM. Mutational analyses suggested that LPS may enter the central cavity laterally, via the interface of the TMD domains of LptF and LptG, and is expelled into the beta-jellyroll-like domains upon ATP binding and hydrolysis by LptB. These studies suggest a mechanism for LPS extraction by LptB2FG that is distinct from those of classical ABC transporters that transport substrates across the IM (Luo et al. 2017). Transport involves a stable association between the inner (LptBFG) and outer (LptDE) membrane components, supporting a mechanism in which lipopolysaccharide molecules are pushed one after the other across a protein bridge (LptCA) that connects the inner and outer membranes (Sherman et al. 2018). The ABC transporter, LptB2FG, which tightly associates with LptC, extracts lipopolysaccharide out of the inner membrane.Li et al. 2019 characterized the structures of LptB2FG and LptB2FGC in nucleotide-free and vanadate-trapped states, using single-particle cryo-electron microscopy. These structures resolve the bound lipopolysaccharide, reveal transporter-lipopolysaccharide interactions with side-chain details and uncover how the capture and extrusion of lipopolysaccharide are coupled to conformational rearrangements of LptB2FGC. LptC inserts its TMS between the two transmembrane domains of LptB2FG, which represents a previously unknown regulatory mechanism for ABC transporters. These results suggest a role for LptC in achieving efficient lipopolysaccharide transport, by coordinating the action of LptB2FG in the inner membrane and Lpt protein interactions in the periplasm (Li et al. 2019). cryo-EM structures of LptB2FG alone and complexed with LptC are known, revealing conformational changes between these states. Two functional transmembrane arginine-containing loops interact with bound AMP-PNP which induces an inward rotation and shift of the transmembrane helices of LptFG and LptC to tighten the cavity, with the closure of two lateral gates, to eventually expel LPS into the bridge (Tang et al. 2019). The ABC transporter, LptB2FGC extracts LPS from the inner membrane and places it onto a periplasmic protein bridge. Lundstedt et al. 2020 showed that residue E86 of LptB is essential for coupling the function of this ATPase to that of its membrane partners, LptFG, at the step where ATP binding drives the closure of the LptB dimer and the collapse of the LPS-binding cavity in LptFG that moves LPS to the Lpt periplasmic bridge consisting of LptC, A and D (from inside to out) and then to the outer membrane insertase, LptE. Defects caused by changing residue E86 are suppressed by mutations altering either the LPS structure or TMSs in LptG. These suppressors fix defects in the coupling helix of LptF, but not of LptG. These observations support a transport mechanism in which the ATP-driven movements of LptB and those of the substrate-binding cavity in LptFG are bi-directionally coordinated through the rigid-body coupling, with LptF's coupling helix being important in coordinating cavity collapse with LptB dimerization (Lundstedt et al. 2020).
Treponema porin, TP0453 of 287 aas. May be involved in ligand transport, altering membrane permeability at acidic pH (4.0 to 5.5) (Luthra et al. 2011). Incubation of the non-lipidated form with lipid vesicles increases their permeability (Hazlett et al. 2005).
The lipoprotein insertase, LolAB, of Gram-negative bacteria
The 36 β-stranded outer membrane porin, CsgG with auxiliary subunits, CsgE and CsgF (Goyal et al. 2014; PMID# 25219853).
Outer membrane phosphate-selective porin OprP (PorP) of 440 aas. Binds and transports a variety of mono, di- and trivalent anions (Benz et al. 1993). An arginine in the pore determines the anion selectivity (Modi et al. 2013). Residues involved in anion affinity and a preference for Pi versus P2 have been identified (Modi et al. 2015). Both monomeric and trimeric OprP are belived to maintain their anion selectivity (Niramitranon et al. 2016). The phosphonic-acid antibiotic fosfomycin is highly permeable through the OprO and OprP channels (Citak et al. 2018).
Pyrophosphate-selective porin OprO (Hancock et al. 1992). The residue basis for the selectivity of P2 over Pi has been determined and involves two residues (Modi et al. 2015). The phosphonic-acid antibiotic fosfomycin is highly permeable through the OprO and OprP channels (Citak et al. 2018).
Attachment GIII (G3P) capsid protein precursor of 434 aa
|1.B.54.1.1||γ-Intimin (Eae protein) (934 aas; Wentzel et al., 2001)||
|1.B.54.1.2||Invasin 985aas (Gal-Mor et al., 2008) (crystal structure of the c-terminal passenger domain has been solved; Hamburger et al., 1999)||
The ZirS/T (ZirS (276 aas)) is the putative exoprotein passenger domain, but it shows no sequence similarity to passenger domains of other Int/Inv family members. ZirT (660 aas) is the outer membrane β-barrel postulated transporter (Gal-Mor et al., 2008).
The β-barrel porin with a superhelical domain containing tetratricopeptide repeats, PgaA or YcdS; exports (deacetylated) poly β-1,6-N-acetyl glucosamine (PGA), a biofilm adhesin that may also play a role in immune evasion (Itoh et al., 2008; Cerca and Jefferson 2008).
Weakly anion-selective OmpA porin. Can exist in two distinct conductance states (Arora et al. 2000). May function in the transport of phenylpropanoids (resveratrol, naringenin and rutin) (Zhou et al. 2014). Three membrane-bound folding intermediates of OmpA were discovered in folding studies with dioleoylphosphatidylcholine bilayers. A highly synchronized mechanism of secondary and tertiary structure formation, applicable to this and other β-barrel membrane proteins has been described (Kleinschmidt 2006).
OmpA of 210 aas. The 3-d structure has been solved by NMR (Renault et al. 2010), and its dynamics have been examined (Renault et al. 2009).
OmpF (OprF) porin. The N-terminal domain has pore activity (Saint et al. 2000). The protein can exist in multiple conformations of variable conductivities (Nestorovich et al. 2006). Factors affecting its one-domain open conformer have been studied by Sugawara et al. (2010). OprF is a complement component C3 receptor (Mishra et al. 2015) and is a target of antibacterial drugs (Maccarini et al. 2017). OprF assumes dual conformations and is involved in solute transport, cell envelope integrity, biofilm formation and pathogenesis (Cassin and Tseng 2019).
OmpATb (ArfA). The central domain (residues 73-220) has been reported to exhibit channel activity (Molle et al., 2006). Its expression is dependent on small single TMS membrane proteins which are encoded in a single operon with it (Veyron-Churlet et al., 2011). The rv0899 gene, encoding OmpATb, is part of an operon (rv0899-rv0901) that is required for fast ammonia secretion, pH neutralization, and growth of M. tuberculosis in acidic environments (Song et al. 2011). Homologues are widespread in bacteria with functions in nitrogen metabolism, adaptation to nutrient poor environments, and/or establishing symbiosis with host organisms (Marassi, 2011). The high resolution 3-d structure is known, revealing two independent domains separated by a proline-rich hinge region.The C-terminal domain (OmpATb(198-326)) revealed a module structurally related to other OmpA-like proteins from Gram-negative bacteria, but the N-terminal domain(73-204), which forms channels in planar lipid bilayers, exhibits a fold, which belongs to the α+β sandwich class fold. It exists in a major monomeric form and a minor oligomeric form yielding rings able to insert into phospholipid membranes (Yang et al. 2011).
Outer membrane porin precursor, OmpX (8 TM β-strands) (NMR structures in lipid bilayers solved (Mahalakshmi et al., 2007; Mahalakshmi and Marassi, 2008)). Expression of the gene is induced by acid or base compared to pH 7 (Stancik et al. 2002).
The attachment inversion locus (Ail) (Bartra et al., 2007). Membrane-bound proteins, Ail and OmpF, are involved in the adsorption of T7-related bacteriophage (Zhao et al. 2013).
Porin of 224 aas and 8 beta strands, TtoA (Estrada Mallarino et al. 2015). The crystal structure is known (3DZM) (Nesper et al. 2008). The 2.8 Å structure reveals a transmembrane 8 stranded β-barrel, an extracellular cation-binding region and an external 5-β stranded sheet (Brosig et al. 2009).
The CarO porin is slightly cation-selective and can be mutated to give rise to imipenem-resistance (Zhu et al. 2019). It is of 243 aas (Siroy et al. 2005) and plays a role in cabapenum resistance (Fonseca et al. 2013) as well as MDR (Yang et al. 2015). It has been implicated in the uptake of ornithine as well as carbapenem antibiotics. Zahn et al. 2015 reported crystal structures of three isoforms of CarO. The structures show a monomeric eight-stranded barrel lacking an open channel. CarO has a substantial extracellular domain resembling a glove that contains all the divergent residues between the different isoforms. A6XB80 is another isoform with 77% identity to the one listed here in TCDB.
PorI (OpmA) porin. Forms channels that allow the passive diffusion of small hydrophilic solutes up to an exclusion limit of about 600 Da. The 3-d structure is known (PDB ID 1PRN).
Blue multi-copper oxidase of 516 aas, CueO. CueO is involved in copper tolerance under aerobic conditions. It features the four typical copper atoms that act as electron transfer (T1) and dioxygen reduction (T2, T3; trinuclear) sites. In addition, it displays a methionine- and histidine-rich insert that includes a helix that blocks physical access to the T1 site (Cortes et al. 2015). It catalyzes oxidation of Mn2+ (Su et al. 2014). Also referred to as copper efflux oxidase (Kataoka et al. 2013).
VDAC1 or VDAC porin of 283 aas, which is > 99% identical to human (P21796) and mouse (60932) VDAC1. Mammals possess three VDACs (VDAC1, 2 and 3) encoded by three genes, but they are all similar in sequence (~60-70% identical) (Messina et al., 2011). The 3-d structure of the human VDAC1 is known (PDB ID 2JK4; Bayrhuber et al. 2008). Reviewed by Shoshan-Barmatz et al. 2015. VDAC1 is found both in mitochondria and the plasma membrane (Lawen et al. 2005) where it may cause cytoplasmic ATP loss.. It may be involved in cancer (Shoshan-Barmatz et al. 2017) and Alzheimer's disease (AD) (Shoshan-Barmatz et al. 2018). Along with its low toxicity profile and high antioxidant activity, the gallic acid derivative, AntiOxBEN3, strongly inhibits calcium-dependent mitochondrial permeability transition pore (mPTP) opening (Teixeira et al. 2018). VDAC dimerization plays a role in mitochondrial metabolic regulation and apoptosis in response to cytosolic acidification during cellular stress, and E73 is involved (Bergdoll et al. 2018). Inhibiting VDAC1 overproduction and plasma membrane insertion in β-cells preserves insulin secretion in diabetes (Zhang et al. 2018). βII and βIII-tubulin, bound to VDAC, regulate VDAC permeability (Puurand et al. 2019). This VDAC porin interacts with carrier precursors arriving in the intermembrane space and recruits TIM22 complexes, thus ensuring efficient transfer of the precursors to the inner membrane translocase (Ellenrieder et al. 2019). A method has been develped to determine the number of VDAC1 channels (and other integral membrane proteins) in nanodiscs under various assembly conditions (Häusler et al. 2020).
Fatty acid outer membrane porin. Gated by high affinity ligand (fatty acid) binding which causes conformational changes in the N-terminus that open up a channel for substrate diffusion (Lepore et al., 2011). May function in the transport of phenylpropanoids (resveratrol, naringenin and rutin) (Zhou et al. 2014).
|1.B.9.2.3||The 14 TMS hydrocarbon porin, TbuX. The crystal structure is known. (3BRY_A) (Hearn et al., 2008).||
Colicin Ia. Residues lining the channel have been identified (Kienker et al. 2008).
Colicin E1 of 522 aas and 1 C-terminal TMS. Ho et al. (2011) suggested a membrane topological model with a circular arrangement of helices 1-7 in a clockwise direction from the extracellular side and membrane interfacial association of helices 1, 6, 7, and 10 around the central transmembrane hairpin formed by helices 8 and 9. ColE1 induces lipid flipping, consistent with the toroidal (proteolipidic) pore model of channel formation (Sobko et al. 2010). The mechanism of channel integration involving the transition of the soluble to membrane-bound form has been presented (Lugo et al. 2016). Colicin E1 uses BtuB as receptor and possibly, the outer membrane TolC protein as the translocator (Cramer et al. 2018). Colicin E1 adopts a closed-channel state at positive transmembrane potentials, correlating with a large tilt angle of alpha-helical TMSs. When the transmembrane potential becomes negative, it inserts into the lipid bilayer with a low tilt angle for the TMSs. Insertion, driven by the negative potential, generates the channel with the open and closed states interconverting reversibly (Su et al. 2019).
Colicin A. The role of the hydrophobic helical hairpin of the pore-forming domain has been elucidated (Bermejo et al. 2013). Acidic conditions promote membrane insertion (Pulagam and Steinhoff 2013).
Colicin B. Its structural stability and interactions have been studied (Ortega et al. 2001).
|1.C.1.3.3||Colicin N (OmpF is the receptor and translocator (Baboolal et al., 2008)).||
|1.C.1.3.4||Colicin S4 (crystal structure known (3FEW_X; Arnold et al., 2009))||
Colicin E2 or E9 (Mosbahi et al., 2002). Colicin E2 is still in contact with its receptor and import machinery when its nuclease domain enters the cytoplasm (Duche, 2007). Colicin E3 is almost identical to Colicin E3 (RNAase). The crystal structure of Colicin E3 with bound BtuB and with the N-terminal translocation (T) domain of E3 and E9 (DNAase) inserted into the OM OmpF porin has been solved (Cramer et al. 2018) revealing: (I) Details of the initial interaction of the colicin central receptor (R)- and N-terminal T-domain with OM receptors/translocators. (II) Features of the translocon include: (a) high-affinity (K d ≈ 10-9 M) binding of the E3 receptor-binding R-domain E3 to BtuB; (b) insertion of disordered colicin N-terminal domain into the OmpF trimer; (c) binding of the N-terminus, documented for colicin E9, to the TolB protein on the periplasmic side of OmpF. Reinsertion of the colicin N-terminus into the second of the three pores in OmpF implies a colicin anchor site on the periplasmic side of OmpF. (III) Studies on the insertion of nuclease colicins into the cytoplasmic compartment imply that translocation proceeds via the C-terminal catalytic domain, proposed here to insert through the unoccupied third pore of the OmpF trimer, consistent with in vitro occlusion of OmpF channels by the isolated E3 C-terminal domain (Cramer et al. 2018).
Pyocin-S2, Pys2 of 689 aas. Causes breakdown of chromosomal DNA as well as complete inhibition of lipid synthesis in sensitive cells. Prevents biofilm formation in vitro and in vivo (Smith et al. 2012). Binds the FpvA receptor (Elfarash et al. 2012). It forms pores though which the toxin enters the cytoplasm (Parret and De Mot 2000).
The alpha-PFT, Haemolysin E, HlyE or ClyA of 536 aas. A peptide derived from the putative transmembrane domain in the tail region of hemolysin E (aas 88-120) assembles in phospholipid membrane and exhibits lytic activity to human red blood cells (Yadav et al., 2009). Residues important for insertion and activity have been identified (Ludwig et al., 2010). An unusual assembly pathway has been proposed (see family description; Fahie et al. 2013). The pore can be blocked by PAMAM dendrimers (Mandal et al. 2016). The C-terminus directs pore formation and function (Sathyanarayana et al. 2016). Similar in structure to Cry6Aa (TC# 1.C.41.2.1) although sequence similarity could not be discerned (Dementiev et al. 2016 and unpublished results). The C-terminal domain is not directly involved in the pore structure, but is not a passive player in pore formation as it plays important roles in mediating the transition through intermediary steps leading to successful pore formation in a membrane (Sathyanarayana et al. 2016). Transmembrane oligomeric intermediates or "arcs" probably form stable proteolipidic complexes consisting of protein arcs with toroidal lipids lining the free edges (Desikan et al. 2017). High-resolution cryo-EM structures revealed that ClyA pore complexes can exist as oligomers of a tridecamer and a tetradecamer, at estimated resolutions of 3.2 Å and 4.3 Å, respectively. The 2.8 A cryo-EM structure of a dodecamer dramatically improves the existing structural model. Structural analysis indicates that protomers from distinct oligomers resemble each other, and neighboring protomers adopt a conserved interaction mode. A stabilized intermediate state of ClyA during the transition process from soluble monomers to pore complexes was identified. Even without the formation of mature pore complexes, ClyA can permeabilize membranes and allow leakage of particles less than ~400 Daltons. In addition, ClyA forms pore complexes in the presence of cholesterol within artificial liposomes (Peng et al. 2019).
The heterokaryon incompatibility prion/amyloid protein, HET-s (Seuring et al., 2012).
The vegetative insecticidal protein, Vip2Ac (462 aas)
Antimicrobial dermcidin, DCD. Based on 3-d structural data, dermcidin forms an architecture of high-conductance transmembrane channels, composed of zinc-connected trimers of antiparallel helix pairs. Molecular dynamics simulations elucidated the unusual membrane permeation pathway for ions and showed adjustment of the pore to various membranes (Song et al. 2013). DCD assembles in solution into a hexameric pre-channel complex before targeting the membrane and integration, the complex follows a deviation of the barrel stave model (Zeth and Sancho-Vaello 2017). The tilt angle and the conductance is determined by the membrane thickness and the cholesterol composition (Song et al. 2019). A soluble 48 residue fragment has been structurally characterized (PDB: 2KSG_A). Membrane interactions and pore formation have been investigated for α-helical AMPs leading to the formulation of three basic mechanistic models: the barrel stave, toroidal, and carpet models. Human cathelicidin (LL-37) and dermcidin (DCD) are α-helical, and their structures have been solved at atomic resolution. DCD assembles in solution into a hexameric pre-channel complex before actual membrane targeting and integration steps occur, and the complex follows a deviation of the barrel stave model (Zeth and Sancho-Vaello 2017).
Bifunctional adenylate cyclase-haemolysin toxin precursor, CyaA. Although homologous, HlyA (1.C.11.1.3) and CyaA exhibit different modes of permeabilization (Fiser and Konopásek 2009). A pore model comprising three alpha2-loop-alpha3 hairpins suggested that Gly530XXGly533XXXGly537 in TMS2 could function in toxin oligomerization (Juntapremjit et al. 2015). Structural integrity of TMSs 1, 2, 3 and 5, but not 4, is important for haemolytic activity, particularly for transmembrane helices 2 and 3 that might form the pore (Powthongchin and Angsuthanasombat 2009). CyaA forms small cation-selective membrane pores that permeabilize cells for potassium efflux, contributing to cytotoxicity of CyaA and eventually provoking colloid-osmotic cell lysis (Wald et al. 2016). The toxin penetrates myeloid phagocytes expressing the complement receptor 3 and delivers into the cytosol its N-terminal adenylate cyclase enzyme domain (~400 residues). In parallel, the ~1300 residue-long RTX hemolysin moiety of CyaA permeabilizes target cell membranes for efflux of cytosolic potassium ions (Svedova et al. 2016). Positively-charged side-chains substituted at positions Gln574 and Glu581 in the pore-lining alpha3 enhance hemolytic activity and ion-channel opening, mimicing the highly-active RTX (repeat-in-toxin) cytolysins (Kurehong et al. 2017). Residues 529 to 549 participate in membrane penetration and pore-forming activity (Roderova et al. 2019). Two distinct conformers of CyaA appear to accomplish its two parallel activities within target cell membranes. The translocating conformer would deliver the N-terminal adenylyl cyclase domain into the cytosol of cells, while the pore precursor conformer would assemble into oligomeric cation-selective pores and permeabilize cellular membrane. Both toxin activities involve a membrane-interacting 'AC-to-Hly-linking segment' (residues 400 to 500). Two clusters of negatively charged residues within this linking segment (Glu419 to Glu432 and Asp445 to Glu448) regulate the balance between the AC domain translocating and pore-forming capacities of CyaA as a function of the calcium concentration (Sukova et al. 2020).
The PNC-37 pore-forming peptide derived from the Mdm-2 binding domain of the p53 tumor-supressor protein which is selectively cytotoxic to cancer cells. The 3-d structure is known from NMR anayses (Sookraj et al. 2010).
Regenerating islet-derived protein 3α, RegIIIα or Reg3A. Also called Proliferation-induced Protein 34, PAP or HIP of 157 aas. It is a C-type intestinal lectin and forms hexameric pores in Gram-positive bacterial membranes. The 3-d x-ray structure is known (Mukherjee et al. 2014). Lipopolysaccharides inhibit pore formation, and hence, Gram-negative bacteria are usually not susceptible to its killing action (Mukherjee et al. 2014).
The adiponectin receptor 1 for ADIPOQ, an essential hormone secreted by adipocytes that regulates glucose and lipid metabolism (Tanabe et al. 2015; Yamauchi et al. 2003.
Required for normal glucose and fat homeostasis and for maintaining a
normal body weight. ADIPOQ-binding activates a signaling cascade that
leads to increased AMPK activity, and ultimately to increased fatty acid
oxidation, increased glucose uptake and decreased gluconeogenesis. Has
high affinity for globular adiponectin and low affinity for full-length
adiponectin. The 3-d structure revealed ceramidase activity for both ADIPOR1 and ADIPOR2; however, the two structures are substantially different (Vasiliauskaité-Brooks et al. 2017). It may function with adiponectin to stimulate cholesterol efflux via ABCA1 (Hafiane et al. 2019).
Moricin of 66 aas and 1 TMS is processed to the active 42 aas peptide. The 3-d solution structure has been solved (1KV4).
Moricin of 67 aas; known to increase permeability of and disrupt cytoplasmic membranes (Hara and Yamakawa 1995).
Moricin of 67 aas
Perfringolysin O, PFO. In the formation of the pore forming toxin, the elongated toxin monomer binds stably to the membrane in an "end-on" orientation, with its long axis approximately perpendicular to the plane of the membrane bilayer (Ramachandran et al. 2005). This orientation is largely retained, even after monomers associate to form an oligomeric prepore complex. The domain 3 (D3) polypeptide segments that ultimately form transmembrane beta-hairpins remain far above the membrane surface in both the membrane-bound monomer and prepore oligomer. Upon pore formation, these segments enter the bilayer, whereas D1 moves to a position that is substantially closer to the membrane. Therefore, the extended D2 beta-structure that connects D1 to membrane-bound D4 appears to bend or otherwise reconfigure during the prepore-to-pore transition of the perfringolysin O oligomer (Ramachandran et al. 2005). The prepore to pore transition has been visualized by electron microscopy (Dang et al. 2005). Phosphatidylcholine in the outer leaflet increases the cholesterol concentration required to induce PFO binding while phosphatidylethanolamine and phosphatidylserine in the inner leaflet of asymmetric vesicles stabilized the formation of a deeply inserted conformation that does not form pores, even though it contains transmembrane segments (Lin and London 2014). This conformation may represent an important intermediate stage in PFO pore formation. Cholesterol recognition, oligomerization, and the conformational changes involved in pore formation have been reviewed (Johnson and Heuck 2014), and the involvement of the D1 domain in structural transitions leading to pore formation has been studied (Kacprzyk-Stokowiec et al. 2014). Interaction of PFO with cholesterol is sufficient to initiate an irreversible sequence of coupled conformational changes that extend throughout the toxin molecule and induce pore formation (Heuck et al. 2007). Once this transmembrane beta-barrel protein is inserted, PFO assembles into pore-forming oligomers containing 30-50 PFO monomers. These form a pore of up to 300 Å, far exceeding the size of most other proteinaceous pores. Decreasing the length of the β-strands causes the pore to shrink (Lin et al. 2015). Site-directed mutagenesis data combined with binding studies performed with different sterols, and molecular modeling are beginning to shed light on the interaction with cholesterol (Savinov and Heuck 2017). Fine-tuning of the stability of beta-strands by Y181 in perfringolysin O directs the prepore to pore transition (Kulma et al. 2019).
Cytohemolysin precursor, HlyA (Vibrio cholerae cytolysin, VCC) is a beta-barrel pore-forming toxin (beta-PFT). A cryo-electron microscopic study revealed low resolution structures for different functional forms (Dutta et al., 2009). Crystal structures of the soluble and transmembrane heptamer reveal common features among disparate pore-forming toxins (De and Olson, 2011). The toxin forms transmembrane heptameric β-barrel channels with two lectin activities on the β-prism and the β-trefoil (Rai et al. 2013). A ring of tryptophan residues forms the narrowest constriction in the transmembrane channel reminiscent of the phenylalanine clamp identified in anthrax protective antigen (Krantz et al., 2005). A single point mutation prevents membrane integration and pore formation (Paul and Chattopadhyay 2012). The deletion of the pre-stem segment does not affect membrane binding and pre-pore oligomer formation, but it critically abrogates the functional pore-forming activity of VCC (Paul and Chattopadhyay 2013). The membrane-bound monomer can not form pores (Rai and Chattopadhyay 2014). VCC can be delivered to host cells via extracellular bacterial vesicles (Elluri et al. 2014). Loops within the membrane-proximal region of VCC play critical roles in determining the functional interactions of the toxin with the membrane lipids that allow pore formation (Rai and Chattopadhyay 2015). VCC may interfer with signalling in the target cell as well as form pores (Khilwani and Chattopadhyay 2015). A functional map of the VCC membrane-binding surface has been published (De et al. 2015). Residues involved in oligomerization have been identified (Rai and Chattopadhyay 2016). The multiple membrane interaction mechanisms of VCC have been reviewed (Kathuria and Chattopadhyay 2018). A model of the transmembrane pore has been presented that accounts for some of its properties (Pantano and Montecucco 2006). An overview of the understanding regarding the membrane interaction mechanisms of VCC and their functional implications for the pore-forming activity of the toxin have been reviewed (Kathuria and Chattopadhyay 2018). The specific cholesterol-binding ability of VCC does not appear to dictate its association with the cholesterol-rich micro-domains on human erythrocytes. Rather, targeting of VCC toward the membrane micro-domains of human erythrocytes possibly acts to facilitate the cholesterol-dependent pore-formation mechanism of the toxin (Cyr 2018).
Vibrio vulnificus hemolysin (VVH-A). Consists of three domains: Hemolysin N (residues 1 - 200), Leukocidin (residues 220 - 480) and Ricin (690 - 600).
Magainin precursor of 333 aas and 1 TMS. 3-d structural determinations and simulations show the oligomeric states, transmembrane helices and tilt angles in the various states of the mature Maganin (Pino-Angeles et al. 2016). Forms stable heterooligomers with PglA (TC# 1.C.16.1.5) at lower concentrations of the two peptides than allows each one alone to form pores in which PglA, rather than magainin 2 forms the pore (Strandberg et al. 2016).
Cecropin A, B and C precursor. Cecropin A and B form pores, but cecropin P1 doesn't. Insertion and activity are dependent on the lipids present. Can be cation- or anion-selective, or non-selective. The negative pole of the dipole is probably inserted into the membrane first (Efimova et al. 2014).
Melittin major precursor (anion selective). Its bacteriocidal activity against Listeria and its cytotoxicity to animal cells have been studied (Wu et al. 2016). In zwitterionic membranes, melittin forms transmembrane toroidal homomeric pores supported by four to eight peptides. Its ability to diffuse freely in a 1,2-dimyristoyl-SN-glycero-3-phosphocholine membrane leads to dynamic pores of vaious diameters with varying molecularity containing from 4 to peptides/channel (Pino-Angeles and Lazaridis 2018).
Defensin 1, 2 and 3 precursor, also called human neutrophil peptide.
|1.C.19.1.5||Defensin-related cryptdin-4 precursor, Crp4 (structure: 2GW9_A) (Cummings and Vanderlick, 2007).||
Pesticide crystal protein Cry4Ba (δ-endotoxin) (1136aas). Cadherin AgCad1 is the receptor for Cry4Ba (Hua et al., 2008). Asn183 in TMS5 is essential for oligomerizatioin of the protein in themidgut membrane of the insect, and therefore for pore formation and toxicity (Likitvivatanavong et al. 2006).
The Cry8Ea1 toxin of 1164 aas. The 2.2-Å crystal structure has been reported (Guo et al. 2009). Cry8Ea1 is specifically toxic to the underground larvae of Holotrichia parallela (Jia et al. 2014).
Class I lantibiotic bacteriocin, Epidermin precursor (has a mersacidin-like Lipid II domain, and forms Lipid II-dependent pores) (Sahl & Bierbaum, 2008). The genetic organization, biosynthesis, modification, excretion, extracellular activation of the modified pre-peptide by proteolytic processing, self-protection of the producer, gene regulation, structure, and mode of actionhave been reviewed (Götz et al. 2014).
|1.C.22.1.6||Plantaricin A precursor||
|1.C.24.1.2||Class IIa bacteriocin Sakacin P precursor||
|1.C.24.1.6||Class IIa bacteriocin Leucocin A precursor||
|1.C.24.1.7||Class IIa bacteriocin Carno(bacterio)cin B2 precursor||
|1.C.25.1.1||Class IIb two peptide bacteriocin Lactococcin G (Oppegard et al., 2007)||
Cyclic bacteriocin, enterocin AS-48, Group I (105 aas; 2 TMSs) (van Belkum et al., 2011) (x-ray structure known (1O82_A))
Cation-selective class IIb two peptide bacteriocin, plantaricin EF (Oppegard et al., 2007). Causes loss of the pmf, K+ release and initiation of apoptosis in Candida species (Sharma and Srivastava 2014). The 3-d structure is known (Fimland et al. 2008).
α-Hemolysin (alpha haemolysin; Hly; Hla; α-toxin). Fragments (13-293 aas) form heptamers like the native full length protein, but a fragment with aas 72-293 formed heptamers, octamers and nonamers. All formed Cl- permeable β-barrel channels (Vécsey-Semjén et al., 2010). The 3-d structure is available (PDB#7AHL). Both symmetry and size of cyclodextrin inhibitors and the toxin pore are important for effective inhibition (Yannakopoulou et al., 2011). Oxoxylin A inhibits hemolysis by hindering self assembly of the hepatmeric pore in which two β-strands are contributed by each subunit (Song et al. 1996; Dong et al. 2013). Applications of pore-forming α-haemolysin include small- and macromolecule-sensing, targeted cancer therapy, and drug delivery (Gurnev and Nestorovich 2014). Sugawara et al. 2015 studied pore formation. Structural comparisons among monomer, prepore and pore revealed a series of motions in which the N-terminal amino latch released upon oligomerization destroys its own key hydrogen bond betweem Asp45 and Try118. This action initiates the protrusion of the prestem. A Y118F mutant and the N-terminal truncated mutant markedly decreased the hemolytic activity, indicating the importance of the key hydrogen bond and the N-terminal amino latch for pore formation. A dynamic molecular mechanism of pore formation was proposed (Sugawara et al. 2015). Release of ATP from cells may occur directly through transmembrane pores formed by α-toxin (Baaske et al. 2016). The amino latch of staphylococcal alpha-hemolysin functions in pore formation via an co-operative interaction between the N terminus and position 217 (Jayasinghe et al. 2006).
PLEKHA7 and other junctional proteins are host factors mediating death by S. aureus alpha-toxin. ADAM10 is docked to junctions by its transmembrane partner Tspan33, whose cytoplasmic C-terminus binds to the WW domain of PLEKHA7 in the presence of PDZD11. ADAM10 is locked at junctions through binding of its cytoplasmic C terminus to afadin. Junctionally clustered ADAM10 supports the efficient formation of stable toxin pores. Disruption of the PLEKHA7-PDZD11 complex inhibits ADAM10 and toxin junctional clustering. This promotes toxin pore removal from the cell surface through an actin- and macropinocytosis-dependent process, resulting in cell recovery from initial injury and survival. Thus, a dock-and-lock molecular mechanism targets ADAM10 to junctions, providing a paradigm for how junctions may regulate transmembrane receptors through their clustering (Shah et al. 2018).
Necrotic enteritis toxin B precursor, NetB (Keyburn et al., 2008)
Two component β-barrel γ-haemolysin, HlgA·HlgB. Tomita et al. (2011) reported that Hlg2 and LukF form a complex, and that Hlg pores form clusters that release hemoglobin from erythrocytes. The crystal structure of this octameric pore (PDB# 3B07; 2QK7) reveals the beta-barrel pore formation mechanism by the two components (Yamashita et al., 2011). Dominant-negative mutant toxins may provide novel therapeutics to combat S. aureus infection (Reyes-Robles et al. 2016). S. aureus beta-barrel pore-forming cytotoxins, including the identification of the toxin receptors on host cells, and their roles in pathogenesis have been reviewed (Reyes-Robles and Torres 2016).
Anion-selective class IIb two peptide bacteriocin, plantaricin J, K (Oppegard et al., 2007). Causes loss of the pmf, K+ loss and initiation of apoptosis in Candida (Sharma and Srivastava 2014).
|1.C.32.1.2||Mastoparan X (INWKGIAAMAKKLL)||
The LL-37 (LL37) peptide (cathelicidin) selectively permeabilizes the membranes of apoptotic human leukocytes, leaving viable cells unaffected (Björstad et al., 2009). It forms transmembrane pores (Lee et al., 2011). It is derived by proteolysis from the cathelin (FALL-39) precursor in granulocytes (Gudmundsson et al. 1996; Li et al. 2016). LL-37 interacts with lipids and shows the formation of oligomers generating fibril-like supramolecular structures on membranes before it assembles into transmembrane pores expressing a modification of the toroidal pore model (Zeth and Sancho-Vaello 2017). Stable transmembrane pore formation occurs at 2.0-10.0 mμM (Lozeau et al. 2018). LL-37 interacts with lipids and forms oligomers generating fibril-like supramolecular structures on membranes before assembling into transmembrane pores with a deviation of the toroidal pore model (Zeth and Sancho-Vaello 2017).
PreIndolicidin (pre-Cathelicidin-4). May function by a carrier mechanism to selectively transport anions (Rokitskaya et al., 2011). The pig (ovine) homologue (SMAP29) is the source from which ovispirin, novispirin and novicidin, which may form torroidal pores, are derived (Sawai et al. 2002).
|1.C.33.1.6||Lipopolysaccharide (LPS) binding protein precursor||
Pro-protegrin-1 (PG-1) (149aas;1 N-terminal TMS) produced by porcine leukocytes. It forms an anion-selective β-sheet toroidal channel of 8 β-hairpins in a consecutive NCCN packing organization, yielding both parallel and antiparallel β-sheets (Jang et al., 2008; Capone et al., 2010). The 3-d structure is known. 97% identical to protegrin-2 (1.C.33.1.1). A model of the protein in Gram-negative bacterial membranes has been proposed (Bolintineanu et al. 2012). Protegrin peptides form octameric pores, and about 100 pores are sufficient to kill E. coli (Bolintineanu et al. 2010). The membrane-bound structure, lipid interactions, and dynamics of the arginine-rich beta-hairpin antimicrobial peptide PG-1 as studied by solid-state NMR are described by Tang and Hong 2009. Protegrin stabilizes partial lipid-forming pores (Prieto et al. 2014). A model of the protegrin-1 pore has been presented, suggesting that permeability of water through a single PG-1 pore is sufficient to cause fast cell death by osmotic lysis (Langham et al. 2008). Possibly, toroidal pore formation is driven by guanidinium-phosphate complexation, where the cationic Arg residues drag the anionic phosphate groups along as they insert into the hydrophobic part of the membrane (Tang et al. 2007). Protegrin-1 is an 18-residue beta-hairpin antimicrobial peptide (AMP) that forms transmembrane beta-barrels in biological membranes. All-atom molecular dynamics simulations of various protegrin-1 oligomers on the membrane surface and in transmembrane topologies indicated that protegrin dimers are stable, whereas trimers and tetramers break down (Lipkin et al. 2017). Tetrameric arcs remained stably inserted in lipid membranes, but the pore water was displaced by lipid molecules. Unsheared protegrin beta-barrels opened into beta-sheets that surrounded stable aqueous pores, whereas tilted barrels with sheared hydrogen bonding patterns were stable in most topologies. A third type of pore consisted of multiple small oligomers surrounding a small, partially lipidic pore. Tachyplesin (TC# 1.C.34.1.1) showed less of a tendency to oligomerize than protegrin: the octameric bundle resulted in small pores surrounded by six peptides as monomers and dimers, with some peptides returning to the membrane surface. Theus, multiple configurations of protegrin oligomers may produce aqueous pores (Lipkin et al. 2017). PG-1 can insert into membranes provided that the external electric potential is large enough to first induce a water column or a pore within the lipid bilayer membrane. The highly charged PG-1 is capable, by itself, of inducing molecular electroporation (Lai and Kaznessis 2018).
Tachyplesin I precursor. It's 3-d pore structure has been examined (Lipkin et al. 2017). Its structure/function and toxicity activities have been reviewed (Edwards et al. 2017).
Cerebroside sulfate activator protein, CSAP or prosaposin (PSAP, GLBA, SAP1) of 524 aas. Saposin A, B, C and D are derived from prosaposin by proteolysis. Saposin-A and C stimulate the hydrolysis of glucosylceramide by beta-glucosylceramidase and galactosylceramide by beta-galactosylceramidase. Saposin-C apparently acts by combining with the enzyme and acidic lipids to form an activated complex, rather than by solubilizing the substrate. Saposin-B stimulates the hydrolysis of galacto-cerebroside sulfate by arylsulfatase A, GM1 gangliosides by β-galactosidase and globotriaosylceramide by α-galactosidase A. Saposin-B forms a solubilizing complex with the substrates of the sphingolipid hydrolases. Saposin-D is a specific sphingomyelin phosphodiesterase activator. Prosaposin behaves as a myelinotrophic and neurotrophic factor; these effects are mediated by its G-protein-coupled receptors, GPR37 and GPR37L1, undergoing ligand-mediated internalization followed by ERK phosphorylation signaling (Hiraiwa et al. 1999).
Antimicrobial natural killer cell lysin, NK-lysin of 129 aas. NK-lysin is involved in the inducible cytotoxicity of T and NK cells (Andersson et al. 1996).
Granulosin of 145 aas and 1 TMS. Functions probably by pore-formation by natural killer (NK) and T lympocyces to combat intracellular parasites, both bacterial and eukaryotic (Dotiwala et al. 2016).
IIITCP protein complex, YopB/YopD (Olsson et al., 2004). TMS2 is essential for function, while TMS1 is partially defective for translocation, pore formation, and signaling (Ryndak et al. 2005). The system forms a multimeric integral membrane complex (Montagner et al., 2011). Mutants have been isoated which show defects in effector translocation and pore formation, and many of these are in a C-terminal domain (Solomon et al. 2015).
IIITCP protein complex, IpaB/IpaC/IpaD. Physical contact with host cells initiates secretion and leads to assembly of a pore, IpaB/IpaC, in the host cell membrane. The active needle tip complex of S. flexneri is composed of a tip protein, IpaD, and the two pore-forming proteins, IpaB and IpaC. The atomic structures of IpaD and a protease-stable coiled-coil fragment in the N-terminal regions of IpaB from S. flexneri and the homologous SipB from Salmonella enterica have been determined (Barta et al. 2012). Structural comparisons revealed similarity to the coiled-coil regions of pore-forming proteins such as colicin Ia (TC# 1.C.1.1.1). Interaction between IpaB and IpaD at the needle tip is key to host cell sensing, orchestration of IpaC secretion and its subsequent assembly at needle tips (Veenendaal et al. 2007). The N-terminus of IpaC is extracellular and the C-terminus is intracellular, and its topology has been studied (Russo et al. 2019).
|1.C.36.3.2||IIITCP protein complex, SipB/SipD of pathogenicity island 1 (SPI1)||
Class IIc bacteriocin, Lactococcin 972 of 91 aas and 1 TM
Sticholysin I (cytolysin ST1; STII; StiII; FraC) (Alvarez et al., 2009). Pore formation goes through a dimer intermediate and then generates the active octamer. Disrupting the key hydrophobic interaction between V60 and F163 (FraC numbering scheme) in the oligomerization interface of FraC, equinatoxin II (EqtII) and sticholysin II (StII) impairs the pore formation activity (Mesa-Galloso et al. 2016). Sticholysin II-mediated cytotoxicity may involve the activation of regulated intracellular responses that anticipates cell death (Soto et al. 2018). Sticholysins represent a prototype of proteins acting through the formation of protein-lipid toroidal pores. Peptides spanning the N-terminus of sticholysins mimic the permeabilizing activity of the full-length toxins (Mesa-Galloso et al. 2019). Phospholipids integrate into the ring of the toroidal pores, promoting their stabilization. Self-association and folding in the membrane determine the mode of action of peptides from the lytic segment of sticholysins (Ros et al. 2019).
Fragaceatoxin C (FraC) of the strawberry anemone (Structure solved to 1.8 Å resolution; It is a crown-shaped nonamer with an external diameter of about 11.0 nm and an internal diameter of approximately 5.0 nm.) Almost identical to Equinatoxin II (1.C.38.1.1) (Mechaly et al., 2011). Fragaceatoxin C (FraC) is an α-barrel pore-forming toxin (PFT). The crystal structures of FraC at four different stages of the lytic mechanism have been determined at 3.1Å resolution, namely the water-soluble state, the monomeric lipid-bound form, an assembly intermediate and the fully assembled transmembrane pore (Tanaka et al. 2015). The structure of the transmembrane pore exhibits a unique architecture composed of both protein and lipids, with some of the lipids lining the pore wall, acting as assembly cofactors. The pore exhibits lateral fenestrations that expose the hydrophobic core of the membrane to the aqueous environment. The incorporation of lipids from the target membrane within the structure of the pore provides a membrane-specific trigger for the activation of this haemolytic toxin. It has been reconstituted in planar lipid bilayers and engineered for DNA analysis. It shows a funnel-shaped geometry that allows tight wrapping around single-stranded DNA (ssDNA), resolving between homopolymeric C, T, and A polynucleotide stretches (Wloka et al. 2016). Despite the 1.2 nm internal constriction in the FraC pore, double-stranded DNA (dsDNA) can translocate through the nanopore at high applied potentials, presumably through deformation of the alpha-helical transmembrane region (Huang et al. 2017). Therefore, FraC nanopores might be useful for DNA sequencing and dsDNA analysis. Pore formation goes through a dimer intermediate and then generates the active octamer. Disrupting the key hydrophobic interaction between V60 and F163 (FraC numbering scheme) in the oligomerization interface of FraC, equinatoxin II (EqtII) and sticholysin II (StII) impairs the pore formation activity (Mesa-Galloso et al. 2016).
MACPF protein. The structure is known (Xu et al., 2010).
Pore-forming, membrane attack, complement component 8, α-polypeptide precursor; C8α-MACPF (structure solved to 2.5 Å resolution; Hadders et al., 2007; Rosado et al., 2007). β-Hairpins in C8α and C9 play a direct role in MAC membrane penetration and pore formation (Weiland et al. 2014). The first TMS of complement component-9 inhibits its own self assembly (Spicer et al. 2018).
Perforin 2; (perforin-2; perforin2;MPEG1) complement component C6; targets phagocytic and some non phagocytic cells (McCormack et al. 2013). Expressed constitutively in phagocytes and inducibly in parenchymal tissue-forming cells. It is a transmembrane protein of cytosolic vesicles, derived from multiple organelles that translocate to and fuse with bacterium containing vesicles. Subsequently, Perforin-2 polymerizes and forms large clusters of 100 Å pores in the bacterial surface with Perforin-2 cleavage products present in the bacteria. Perforin-2 is also required for the bactericidal activity of reactive oxygen and nitrogen species as well as hydrolytic enzymes (McCormack et al. 2015). Perforin-2 exists in membrane-bound (P2a) and secretory (P2b) isoforms, both present in human macrophages.P2a promotes fusion of vesicles with lysosomes (Xiong et al. 2017).
and may play important roles in immune defense.
Chain A, MACPF perforin-like protein, Plu-MACPF (structure solved to 2.0 Å resolution; Rosado et al., 2007).
Aerolysin (β-hemolysin; cytolytic enterotoxin) precursor (Parker et al., 1994). Upon transition from the prepore to pore, the aerolysin heptamer shows a unique concerted swirling movement, accompanied by a vertical collapse of the complex, ultimately leading to the insertion of a transmembrane beta-barrel (Degiacomi et al. 2013). Multiple conformational states lead to rotation of the core lysin to unleash the membrane spanning regions (Whisstock and Dunstone 2013). Monomer activation, dependent on proteolysis, is the rate-limiting step for pore formation (Bischofberger et al. 2016). Cryo-electron microscopy structures of three conformational intermediates and the final aerolysin pore provide insight into the conformational changes that allow pore formation. The structures reveal a protein fold consisting of two concentric beta-barrels, tightly kept together by hydrophobic interactions. This fold suggests a basis for the prion-like ultrastability of aerolysin pore and its stoichiometry (Iacovache et al. 2016).
Parasporin-2 β-toxin (crystal structures are known) (Akiba et al., 2009; Akiba and Okumura 2016).
Bacterial permeability inducing protein, BPIP precursor, of 487 aas and 1 N-terminal TMS.
Bacillus anthracis protective antigen (PA). Many cationic compounds inhibit in nM - mM concentration ranges (Yamini et al. 2016). Both symmetry and size of cyclodextrin inhibitors and the toxin pore are important for effective inhibition (Yannakopoulou et al., 2011). A cryo electron microscopic structure of the anthrax protective antigen translocon and the N-terminal domain of anthrax lethal factor inserted into a nanodisc model lipid bilayer has been solved revealing a cap, a narrow stalk and a transmembrane channel (Gogol et al. 2013). Poly(amindo)amine (PAMAM) dentrimers block activity (Förstner et al. 2014). The 3-d structure of PA, showing the channel and the φ-clamp, and providing information about the multi-step mechanism by which low pH is sensed and the membrane-spanning channel is formed has been published (Jiang et al. 2015). The export of the lethal factor and edema factor from the endosome into the host cytosol is dependent on the proton motive force (pmf) (Krantz et al. 2006; Colby and Krantz 2015). Translocation of anthrax toxin's lethal factor is initiated by entry of its N terminus into the protective antigen channel (Zhang et al. 2004).
C2II channel-forming toxin component. Channel-formation is inhibited by azolopyridinium salts (Bronnhuber et al. 2014).
Lysenin of 297 aas and 1 N-terminal TMS, a sphingomyelin-specific pore-forming toxin from earthworms; causes contraction of rat vascular smooth muscle. (Sekizawa et al., 1997; Shogomori and Kobayashi, 2007). Trp-20 is required for cation selective channel assembly (Kwiatkowska et al., 2007). Adenosine phosphates control the activity of lysenin channels inserted into planar lipid membranes with respect to their macroscopic conductance and voltage-induced gating. Addition of ATP, ADP, or AMP decreased the macroscopic conductance of lysenin channels in a concentration-dependent manner, with ATP being the most potent inhibitor and AMP the least (Bryant et al. 2016). lysenin can specifically interact with sphingomyelin, and may confer innate immunity against parasites by attacking the membranes of the parasites to form pores (Pang et al. 2019). Upon binding to sphingomyelin (SM)-containing membranes, lysenin undergoes a series of structural changes promoting the conversion of water-soluble monomers into oligomers, leading to its insertion into the membrane and the 2-step formation of a lytic beta-barrel pore (Kulma et al. 2019). Structural stabilization of the lysenin prepore starts at the site of initial interaction with the lipid membrane and is transmitted to the twisted beta-sheet of the N-terminal domain (Kulma et al. 2019). 3-d structures are available (PDB# 5EC5; 3ZXD; 3ZX7). The beta pore-forming toxins (beta-PFTs) are cytotoxic proteins produced as soluble monomers, which cluster and oligomerize at the membrane of the target host cells. Their initial oligomeric state, the prepore, is not cytotoxic. The beta-PFTs undergo a large structural transition to a second oligomeric state, the pore, which pierces the membrane of the host cell and is cytotoxic. Munguira et al. 2019 described the mechanism by which the rates of formation of the transmembrane pores correlate with the local levels of crowding for the beta-PFT lysenin.
|1.C.44.1.1||β-purothionin (A-I) precursor||
|1.C.44.1.2||Viscotoxin B precursor||
|1.C.45.1.1||Antifungal protein 1, RsAFP1 prercursor||
|1.C.45.1.2||Flower-specific g-thionin precursor||
The antifungal lentil seed defensin, Lc-def (47aas plus of 27aa leader peptide) (Finkina et al., 2008)
|1.C.46.1.1||CNP precursor protein (CNP-22 and CNP-29)||
L-Plectasin (40aas, 1 TMS); precursor (90aas, 2 TMSs). 3-d structure known (3E7R_L; 1ZFUA) (Mygind et al., 2005; Zhu, 2008) (43% identical to 1.C.47.1.1).
|1.C.48.1.1||Major prion protein precursor PrP (yielding peptide PrP[106-126])||
Calcitonin gene regulatory peptide I precursor, CGRPI of 128 aas and 1 - 3 TMSs. CGRP induces vasodilation, dilating a variety of vessels including the coronary, cerebral and systemic vasculature. Its abundance in the CNS points toward a neurotransmitter or neuromodulator role (Kitamura et al. 1992). It also elevates platelet cAMP and elevates calcium while polarizing the membrane potential by both cAMP-independent and -dependent mechanisms (Burns et al. 2004).
Alzheimer''s disease amyloid β-protein (amino acids 1-42) (Abeta protein or AβP or Aβ42). Aβ pores may consist of tetrameric and hexameric beta-sheet subunits (Strodel et al. 2010). Residues 22 - 35 in the peptide binds cholesterol to form Ca2+-permeable pores (Di Scala et al. 2014). Cholesterol promotes the insertion of Abeta in the plasma membrane, induces alpha-helical structure formation, and forces the peptide to adopt a tilted topology that favours oligomerization. Bexarotene, an amphipathic drug for the treatment of neurodegenerative diseases, competes with cholesterol for binding to Abeta and prevents oligomeric channel formation (Di Scala et al. 2014). The beta-amyloid protein is involved in the activation of the nAChRalpha7 receptor (Hassan et al. 2019).
The Alzheimer’s disease amyloid β-protein (Aβpeptide; precursor: App, γ-secretase) (42aas) (3-d structure is known from NMR spectroscopy (1Z0Q_A; Jang et al., 2007; Zheng et al., 2008)). This peptide is derived from the amyloid βA4 protein isoform f (NP_001129602)) which forms variable oligomeric toxic pores leading to cytosolic calcium elevation and Alzheimer's disease (Demuro et al., 2011). The monomer of Ass1-42 normally activates type-1 insulin-like growth factor receptors and enhances glucose uptake in neurons and peripheral cells by promoting the translocation of the Glut3 glucose transporter from the cytosol to the plasma membrane (Giuffrida et al. 2015). At nanomolar concentrations, APPsα is an allosteric activator of α7-nAcChR (see TC family 1.A.9), mediated by the C-terminal 16 aas (CTα16) (Korte 2019).
Shiga toxin B Chain (StxB; verotoxin B chain) precursor, ST-B
Cytotoxin B, TcdB. The minimal pore-forming region is within amino acid residues 830 and 990 including glutamate-970 and glutamate-976. These two residues are essential for pore formation (Genisyuerek et al., 2011). Other residues important for toxicity have been identified (Zhang et al. 2014). Residues in the translocation domain of TcdB that form the pore and function in toxin translocation have been identified (Hamza et al. 2016).
|1.C.57.3.1||Pasteurella multocida toxin (PMT); dermonecrotic toxin (DMT); mitogenic toxin (ToxA) (Baldwin et al., 2004)||
CPE (Clostridium perfringens Enterotoxin) has been used for suicide gene therapy for selective treatment of claudin-3-and-4-overexpressing tumors (Walther et al., 2011). It can be used as an oncoleaking/tumor eradication agent as this pore-forming protein exerts specific and rapid toxicity towards claudin-3- and -4-overexpressing cancers (Pahle et al. 2015). The crystal structure of Clostridium perfringens enterotoxin displays features of beta-pore-forming toxins (Kitadokoro et al., 2011). The N-terminal region (nCPE) mediates the cytotoxic effect through pore formation in the plasma membrane of the mammalian host cell. The C-terminal region (cCPE) binds to the second extracellular loop of a subset of claudins, Claudin-3 and claudin-4, with high affinity (Veshnyakova et al., 2010). cCPE is not cytotoxic but is a potent modulator of tight junctions. The toxin forms highly cation-selective channels in lipid bilayers (Benz and Popoff 2018). Mepacrine inhibits CPE-induced electrophysiology effects in artificial lipid bilayers lacking CPE receptors by blocking subunit oligomerization and pore formation (Freedman et al. 2017).
Neurotoxin of 623 aa
The β-barrel pore-forming toxin (PFP), Monalysin. The soluble monomer is cleaved to yield oligomeric pores. The structure of a cleaved form lacking the transmembrane domain has been solved by x-ray crystalography and cryo-EM (PDB#4MJT; Leone et al. 2015). The structure displays an elongated shape, resembling those of beta-pore-forming toxins such as aerolysin, but it lacks the receptor binding domain. Pro-monalysin forms a stable doughnut-like 18-mer complex composed of two disk-shaped nonamers held together by N-terminal swapping of the pro-peptides. This is in contrast with the monomeric pro-form of the other beta-PFTs that are receptor-dependent for membrane interaction. The membrane-spanning region of pro-monalysin is fully buried in the center of the doughnut, suggesting that upon pro-peptide cleavage, the two disk-shaped nonamers can - and have to - dissociate to leave the transmembrane segments free to deploy and lead to pore formation. In contrast with other toxins, the delivery of 18 subunits at once, nearby the cell surface, may be used to by-pass the requirement for a receptor-dependent concentration to reach the threshold for oligomerization into the pore-forming complex (Leone et al. 2015).
|1.C.64.1.1||Fst peptide toxin||
|1.C.71.1.1||The Cyt1Aa δ endotoxin||
The Cyt2Aa δ endotoxin of 259 aas. Cyt2Aa2 binds and aggregates on the lipid membrane leading to the formation of non-specific holes and disruption of the cell membrane (Tharad et al. 2016). The crystal structure is available (PDB 3RON).
|1.C.71.2.1||The volvatoxin A2 precursor||
The Subtilase cytotoxin, SubAB. Pentameric SubB, but not SubA, is homologous to ArtB of Salmonella enterica. SubA (AB5 subtilase) cytotoxin inactivates the endoplasmic reticulum chaperone, BiP (Paton et al., 2006; Beddoe et al., 2010).
Pore-forming exotoxin A (chain A; ExlA) (Rasper and Merrill 1994; Méré et al., 2005). Pore-formation has been demonstrated (Zalman and Wisnieski 1985). Secretion depends on ExlB, a Two Partner Secretion (TPS; TC# 1.B.20) system, as well as type IV pili. The protein has three domains: an N-terminal hemolyin domain, a central RGD motif domain, and a C-terminal domain required for cell lysis. Pore-formation precedes lysis (Basso et al. 2017). ExlA triggers cadherin cleavage by promoting calcium influx which activates ADAM10 for proteolysis (Reboud et al. 2017).
The cholix toxin. The NAD-dependent ADP-ribosyltransferase (ADPRT) catalyzes transfer of the ADP-ribosyl moiety of oxidized NAD onto eukaryotic elongation factor 2 (eEF-2), thus arresting protein synthesis. It may use the eukaryotic pro-low-density lipoprotein receptor-related protein 1 (LRP1) to enter mouse cells, Cholix toxin shares structural and functional properties with Pseudomonas aeruginosa exotoxin A and Corynebacterium diphtheriae diphtheria toxin (Lugo and Merrill 2015).
|1.C.74.1.2||α-bungarotoxin isoform A31 (α-BTX A31) (blocks activity of the nicotinic acetylcholine receptor (TC #1.A.9)||
Bucain of 65 aas
3 finger muscarinic toxin, Mt1 of 66 aas. Shows a non-competitive interaction with adrenergic and muscarinic receptors. Binds to alpha-2b (ADRA2B) (IC50=2.3 nM), alpha-1a (ADRA1A), alpha-1b (ADRA1B), and alpha-2c (ADRA2C) adrenergic receptors. Reversibly binds to M1 (CHRM1) muscarinic acetylcholine receptors, probably by interacting with the orthosteric site.
α-elapitoxin 2a, Nno2a of 73 aas
Maculatin 1.1 (21 aas); similar to caerin (1.C.52.1.9) (Fernandez et al., 2008; Mechler et al., 2007). Maculatins self assemble in parallel to form pores in phospholipid bilayers (Sani et al. 2020).
α-synuclein (140 aas). In addition to β-amyloid, the cellular prion protein, PrPC binds α-synuclein, which is responsible for neurodegenerative synucleopathies (Urrea et al. 2017). β-barrel channels such as α-hemolysin may serve as sensitive probes of α-synuclein (α-syn) interactions with membranes as well as model systems for studies of channel-assisted protein transport (Gurnev et al. 2014). α-synuclein interacts with membranes to affect Ca2+ signalling, and the oligomeric β-sheet-rich α-synuclein leads to Ca2+ dysregulation and Ca2+-dependent cell death (Angelova et al. 2016).
|1.C.78.1.2||Crystal protein, CryET33||
CRY51Aa insecticidal aerolysin-type β-pore-forming toxin of 309 aas. The crystal structure is available (Xu et al. 2015). Cry35 and Cry51 belong to protein families (Toxin_10, ETX_MTX2) sharing a common β-pore forming structure and function with known mammalian toxins such as epsilon toxin (ETX) (Moar et al. 2016).
Botulinum neurotoxin types A-G. Poly(amindo)amine (PAMAM) detrimers block activity (Förstner et al. 2014). BoNTs inhibit synaptic exocytosis; intoxication requires the di-chain protein to undergo conformational changes in response to pH and redox gradients across the endosomal membrane with consequent formation of a protein-conducting channel by the heavy chain (HC) that translocates the light chain (LC) protease into the cytosol, colocalizing it with the substrate SNARE proteins (Montal 2009).
Clostridium botulinum neurotoxin type E (3d structure known (Kumaran et al., 2009))
Arenicin-1 precursor (202 aas). The processed pore-forming β-hairpin antimicrobial peptide corresponds to residues 182-202 (Andrä et al., 2008; Shenkarev et al., 2011). Low-conductivity pores were detected in the phosphatidylethanolamine-containing lipids and high-conductivity pores in anionic lipids. The measured conductivity levels agreed with the model in which arenicin antimicrobial activity was mediated by the formation of toroidal pores assembled of two, three, or four β-structural peptide dimers and lipid molecules (Shenkarev et al., 2011).
Leakage-promoting cyclic peptide, Subtilosin (43aas)
β-defensin-3 of 67 aas and 1 N-terminal TMS. Canine BD103 (van Damme et al. 2009) is 79% identical.
|1.C.89.1.1||β-neoendorphin-dynorphin precursor (Proenkephalin B; Preprodynorphin)||
|1.C.89.1.2||Proenkephalin A (264aa precursor of opioid peptides)||
Cyclic bacteriocin, Group I, carnocyclin, (CdlA; 64 aas; 1 TMS) (van Belkum et al., 2011) (3-d solution structure: 2KJF_A; forms anion selective pores; Similar to As-48 (1.C.28.1.1)) (Martin-Visscher et al., 2009)
The tetrameric Stefin B pore-forming protein (98aas); structure known (20CT_A)
|1.C.92.1.1||C-reactive protein 1.1 precursor, CRP1.1||
|1.C.92.1.2||Serum amyloid P component precursor, SAP (223aas)||
Uncharacterized protein, Thuricin CD or Trnβ, of 49 aas and 1 TMS.
ThuricinCD or Trnα of 47 aas and 1 TMS
Pore-forming ESAT-6 (EsxA) (95 aas) (Nuñez-Garcia et al. 2018). Secreted from the bacterial cytoplasm via a ESX protein secretion system (Type VII; TC# 3.A.24.5.1).
The haemolytic lectin, CEL-III (Uchida et al. 2004). CEL-III heptamerizes via a large structural transition from alpha-helices to a beta-barrel during the transmembrane pore-formation process (Unno et al. 2014).
Pleurotolysin A/B pore-forming toxin. Pleurotolysin A (PlyA; also called ostreolysin A, OlyA) binds first in a sphingomyelin-dependent process; Pleurotolysin B (PlyS) binds to A in the membrane and inserts (Kondos et al., 2011). The binary cytolytic pore-forming complex forms non-selective ion conducting pores of variable size (Schlumberger et al. 2013) to promote fruiting (Ota et al. 2014). Conformational changes accompanying pore formation have been reported (Lukoyanova et al. 2015). In these systems, the aegerolysin-like proteins provide the membrane cholesterol/sphingomyelin selectivity and recruit oligomerised pleurotolysin B molecules, to create a membrane-inserted pore complex. The resulting protein structure has been imaged with electron microscopy, and it has a 13-meric rosette-like structure, with a central lumen that is ~4-5 nm in diameter. The opened transmembrane pore is non-selectively permeable for ions and smaller neutral solutes, and is a cause of cytolysis of a colloid-osmotic type (Ota et al. 2014).
The heterotrimeric CDT, CdtA/B/C toxin complex. CdtA and CdtC may form a heterodimeric complex required for CdtB delivery. Localized to the cell outer membrane. Contains a ricin B-type lectin domain (Smith and Bayles 2006).
The SNARE fusion complex, fusing neurotransmitter vesicles with the presynaptic membrane. Ca2+ acts on the synaptic vesicle synaptotagmin1 (synaptotagmin I; SytI, Syt1, SSVP65, SYT) to trigger rapid exocytosis (Chapman, 2008). Syt1 is a major Ca2+ sensor for fast neurotransmitter release. It contains tandem Ca2+-binding C2 domains (C2AB), a single transmembrane α-helix and a highly charged 60-residue- long linker in between. The linker region of Syt1 is essential for its two signature functions: Ca2+-independent vesicle docking and Ca2+-dependent fusion pore opening. The linker contains the basic-amino acid-rich N-terminal region and the acidic amino acid-rich C-terminal region (Lai et al. 2013). The intrinsically disordered region between Syt I's transmembrane helix and the first C2 domain interats with vesicular lipids and modulates Ca2+ binding to C2 (Fealey et al. 2016). t-SNARE and v-SNARE interact in their C-terminal TMSs to promote pore opening (Wu et al. 2016). Both sides of a trans-SNARE complex can drive pore opening suggesting an indentation model in which multiple SNARE C-termini cooperate in opening the fusion pore by locally deforming the inner leaflets (D'Agostino et al. 2016). The TMSs of SNARE proteins regulate the fusion process (Wu et al. 2017). The cysteine-rich domain of SNAP-23 regulates its membrane association and exocytosis from mast cells (Agarwal et al. 2019). Snc1 is trafficked between the endosomal system and the Golgi apparatus via multiple pathways, providing evidence for protein quality control surveillance of a SNARE protein in the endo-vacuolar system (Ma and Burd 2019).
Yeast vacuolar snare complex including the vesicle-associated membrane protein 2 (Snc2p; 115aas; 1-C-terminal TMS) (Chernomordik et al., 2005), the vacuole morphogenesis protein, Vam3 (PTH1) of 283 aas, the vacuolar v-snare, Nyv1 of 253 aas, and the t-snare, Vti1 of 217 aas. Considering these last three proteins, SNARE TMSs serve as non-specific membrane anchors in vacuole fusion, but fusion requires the SNARE complexes in the plasma and vacuolar membranes. Lipid-anchored Vti1 was fully active, lipid-anchored Nyv1 (R-SNARE) permitted the fusion reaction to proceed up to hemifusion, but lipid-anchored Vam3 interfered with fusion before hemifusion. Vam7 (a soluble SNARE; 316 aas) and Sec18 (758 aas) remodel SNARE compexes to allow lipd-anchored R-SNARE (NYV1, 253 aas), acting with Q-SNARE (VTS1; 523 aas), to support vacuole fusion (Jun et al. 2007).Thus, these proteins have non-specific membrane anchors, but each of these proteins makes different contributions to the hemifusion intermediate and opening of the fusion pore (Semenov et al. 2014). The 181-198 region of Qa-snare, immediately upstream of the SNARE heptad-repeat domain, is required for normal fusion activity with HOPS. This region is needed for normal SNARE complex assembly (Song and Wickner 2017). Sec17 and Sec18 act twice in the fusion cycle, binding to trans-SNARE complexes to accelerate fusion, and then to hydrolyze ATP to disassemble cis-SNARE complexes (Song et al. 2017).
The mouse synaptobrevin 2 (syb2)/VAMP2/Syntaxin (Syx)/SNAP-25 complex involved in vesicle fusion pore formation (Chang et al. 2015). The synaptobrevin juxtamembrane regions plus the TMS may catalyze pore formation by forming a membrane-spanning complex that increases curvature stress at the circumference of the hemifused diaphragm of the prepore intermediate state (Tarafdar et al. 2015). The TMS of VAMP2 plays a critical role membrane fusion, and the structural mobility provided by the central small amino acids is crucial for exocytosis by influencing the molecular re-arrangements of the lipid membrane that are necessary for fusion pore opening and expansion (Hastoy et al. 2017). SNARE TMSs may function as parts of the fusion pores during Ca2+-triggered exocytosis for release of both neurotransmitters and hormones (Chiang et al. 2018). The intracellular periodontal pathogen, P. gingivalis, exploits a recycling pathway involving VAMP2 to exit from infected cells (Takeuchi et al. 2016).
Dysferlin/Caveolin 3/MG53 (TRIM72) complex. Mediates vesicle fusion and membrane repair in muscle cells (Fuson et al. 2014). Dysferlin (DysF; Fer1L1) belongs to the Ferlin family. A deficiency of dysferlin, which binds lipids in a Ca2+-dependent process, causes vesicle accumulation near membrane lesions (Roostalu and Strähle 2012).
The herpes envelope glycoprotein class III membrane fusion system including glycoproteins gB, gD, gH and gL. There are two fusion peptides of 8 aas each that form a bipartite system (Apellániz et al. 2014; Feng and Jia 2016).
The poxvirus entry protein complex of Vaccinia virus WR. F9 and L1 are homologous, and G9 and J5 may be homologous as well.
Avian leukosis virus (RSV) envelope glycoprotein, gp95 or EnvA (606aas; 2 TMSs). Mediates pore formation preceded by a relatively stable hemifusion-like intermediate (Jha et al., 2011). A shorter version is of 138 aas and has two TMSs at the N- and C-termini. It's acc# is H7CEB0. The fusion peptide is 28 aas with a single TMS (Apellániz et al. 2014).
Ebola virus glycoportein 2 of 676 aas. The NMR structure of the internal fusion loop of 54 aas has been solved (2LCY) The fusion peptide is 17 aas long (Apellániz et al. 2014). The GP2 protein also encoedes the GP2-δ peptide of 40 aas which is a viroporin (He et al. 2017). This nonstructural polypeptide, called the delta peptide, is produced in abundance during Ebola virus infection. Full length and conserved C-terminal delta peptide fragments permeabilize the plasma membranes of nucleated cells, increase ion permeability across confluent cell monolayers and permeabilize synthetic lipid bilayers. Permeabilization activity is dependent on the disulfide bond between the two conserved cysteines. The conserved C-terminal portion of the peptide is biochemically stable in human serum, and most serum-stable fragments have full activity (He et al. 2017).
Full virion envolope spike glycoprotein, GP1.2, of 681 aas. The intervan fusion peptide is 15 aas long (Apellániz et al. 2014).
The p15 fusion-associated small transmembrane (FAST) protein is a nonstructural viral protein that induces cell-cell fusion and syncytium formation (Top et al. 2012). The small, myristoylated N-terminal ectodomain of p15 lacks any of the defining features of a typical viral fusion protein. NMR and CD spectroscopy indicated that this small fusion module (residues 68 - 87) comprises a left-handed polyproline type II (PPII) helix flanked by small, unstructured N- and C-termini (PDB# 2MNS_A). Individual prolines in the 6-residue proline-rich motif are tolerant to alanine substitutions, but multiple substitutions that disrupt the PPII helix eliminate cell-cell fusion activity. A synthetic p15 ectodomain peptide induces lipid mixing between liposomes. Lipid mixing, liposome aggregation, and stable peptide-membrane interactions are all dependent on both the N-terminal myristate and the presence of the PPII helix. A model for the mechanism of action of this viral fusion peptide, whereby the N-terminal myristate mediates initial, reversible peptide-membrane binding that is stabilized by subsequent amino acid-membrane interactions. These interactions induce a biphasic membrane fusion reaction, with peptide-induced liposome aggregation representing a distinct, rate-limiting event that precedes membrane merger. The PPII helix may function to force solvent exposure of hydrophobic amino acid side chains in the regions flanking the helix to promote membrane binding, apposition, and fusion (Top et al. 2012). A fusion-inducing lipid packing sensor (FLiPS) in the cytosolic endodomain in the p15 fusion-associated small transmembrane (FAST) protein is essential for pore formation during cell-cell fusion and syncytiogenesis (Read et al. 2015). The Myristoylated Polyproline Type Ii Helix Protein of 22 aas (residues 68 - 87 in P15) functions as a fusion peptide during cell-cell membrane fusion. The 3-d structure is known (PDB# 2LKW).
Influenza C virus hemagglutinin-fusion pore-forming protein of 655 aas and 4 TMSS, one N-terminal and three C-terminal but separated by about 100 residues. Pore formation is blocked by human interferon-induced transmembrane proteins such as IFM3 (Q01628) (Desai et al. 2014). The only spike of influenza C virus, the hemagglutinin-esterase-fusion glycoprotein (HEF) combines receptor binding, receptor hydrolysis and membrane fusion activities in a single protein. Like other hemagglutinating glycoproteins of influenza viruses, HEF is S-acylated, but only with stearic acid at a single cysteine located at the cytosol-facing end of the transmembrane region. S-acylation is essential for replication of influenza viruses A, B and C by affecting budding and/or membrane fusion (Wang et al. 2016).
The major envelope protein, GP64, of 512 aas and 2 TMSs (N- and C-terminal). The two fusion peptides of this type III bipartite system are residues 75 - 88 and 145 - 160 (Apellániz et al. 2014). The GP64 transmembrane domain is essential for GP64 trafficking, membrane fusion, virion budding, and virus infectivity but could be replaced only by transmembrane domains from related viral membrane proteins (Li and Blissard 2008).
Glycoprotein 160, GP160, Env protein of 854 aas. This is the intact protein from which GP41 is derived by proteolysis.
The rotavirus A membrane fusion protein complex including VP5 and 8, derived by proteolysis of VP4, as well as VP7, VP6 and VP2. VP5, 8 and 7 may play primary roles while VP6 and 2 play secondary roles (Gilbert and Greenberg 1998; Golantsova et al. 2004; Settembre et al. 2011; Elaid et al. 2014). These proteins from different viral strains may be very divergent in sequence.
The respiratory syncytial virus (RSV) fusion (F) glycoprotein. The crystal strcuture is available (McLellan et al. 2013). The protein has at least 3 conformational states: pre-fusion native state, pre-hairpin intermediate state, and post-fusion hairpin state. During viral and plasma cell membrane fusion, the heptad repeat (HR) regions assume a trimer-of-hairpins structure, positioning the fusion peptide in close proximity to the C-terminal region of the ectodomain. The formation of this structure appears to drive apposition and subsequent fusion of viral and plasma cell membranes which leads to delivery of the nucleocapsid into the cytoplasm. Fusion is pH independent and occurs directly at the outer cell membrane. The trimer of F1-F2 (protein F) interacts with glycoprotein G at the virion surface. Upon binding of G to heparan sulfate, the hydrophobic fusion peptide is unmasked and interacts with the cellular membrane, inducing the fusion between host cell and virion membranes. RSV fusion protein is able to interact directly with heparan sulfate and therefore actively participates in virus attachment.
Tick-borne encephalitis virus (TBEV) (Class II) polyprotein of 3414 aas. Residues 281 - 776 include the envelop protein that includes the viral fusion protein (Zhang et al. 2017).
Polyprotein (3391aas) (includes the membrane fusion protein, envelope protein E (495aas; 38% identical to residues 282-774 in 1.G.3.1.1) (Liao et al., 2010)). The fusion peptides are residues 98 - 113 in V7SFC4 and residues 378 - 393 in P14340 (Apellániz et al. 2014).
The Semliki Forest Virus (SFV) (Class II) Structural polyprotein (1253 aas; E1=816-1253 E2=334-774). The fusion peptide is residues 895 - 913 (Apellániz et al. 2014). The 6K viroporin transports monovalent cations and Ca2+ (Hyser and Estes 2015).
|1.G.4.1.3||The Barmah Forest virus 6K protein (58 aas; present within the viral structural polyprotein (P89946))||
|1.G.5.1.1||The Vesicular Stomatitis Virus (VSV) Glycoprotein G (423 aas)||
Pre-glycoprotein polyprotein (precursor), GPC (York and Nunberg, 2009).
Claudin 2 (Claudin-2; CLDN2) (forms narrow, fluid filled, water-permeable cation-selective paracellular pores) (Angelow et al., 2008; Yu et al., 2009). It is a dimer in a high molecular weight protein complex (Van Itallie et al. 2011; Krug et al. 2014). Transports Na+, K+, Ca2+ smal organic molecules and water through the paracellular channel (Fromm et al. 2017). Site-specific distributions of claudin-2- and claudin-15-based paracellular channels drive their organ-specific functions in the liver, kidney, and intestine (Tanaka et al. 2017). Disruption of the gastrointestinal epithelial barrier is a hallmark of chronic inflammatory bowel diseases (IBDs), and in the intestines of patients with IBDs, the expression of CLDN2 is upregulated (Takigawa et al. 2017). Leu increases Ca2+ flux through cellular redistribution of Cldn-2 to the tight junction membrane (Gaffney-Stomberg et al. 2018).
Claudin-15 of 227 aas and 4 TMSs, Cldn15. Suzuki et al. 2013 reported the crystal structure of mouse claudin-15 at a resolution of 2.4 angstroms. The structure revealed a characteristic β-sheet fold consisting of two extracellular segments anchored to a transmembrane four-helix bundle by a consensus motif. Potential paracellular pathways with distinctive charges on the extracellular surface provided insight into the molecular basis of ion homeostasis across tight junctions. Site-specific distributions of claudin-2- and claudin-15-based paracellular channels drive their organ-specific functions in the liver, kidney, and intestine (Tanaka et al. 2017). A model of the claudin-15-based paracellular channel has been presented (Alberini et al. 2017).
Claudin 4 (209aas) forms paracellular chloride channels in the kidney
collecting duct and requires Claudin 8 for tight junctions localization (Hou et al., 2010).
NPC (Tran and Wente, 2006). The structure of the NPC core (400kD) has been determined at 7.4 Å resolution revealing a curved Y-shaped architecture with the coat nucleoporin interactions forming the central ""triskeleton"". 32 copies of the coat neucloporin complex (CNC) structure dock into the cryoelectron tomographic reconstruction of the assembled human NPC, thus accountng for ~16 MDa of it's mass (Stuwe et al. 2015). Import of integral membrane proteins (mono- and polytopic) into the the inner nuclear membrane occurs by an active, transport factor-dependent process (Laba et al. 2015). Ndc1 and Pom52 are partially redundant NPC components that are essential for proper assembly of the NPC. The absence of Ndc1p and Pom152p results in aberrant pores that have enlarged diameters and lack proteinaceous material, leading to increased diffusion between the cytoplasm and the nucleus (Madrid et al. 2006).