Leech innexin, Inx2 (Kandarian et al. 2012; Firme et al. 2012)

Animals

Inx2 of Hirudo verbana

Duplicated innexin of 801 aas and 8 TMSs.

Innexin of Ascaris suum

Duplicated innexin protein of 813 aas and 8 TMSs.

Duplicated innexin of Trichinella spiralis (Trichina worm)

Innexin2, Inx2 of 359 aas and 4 TMSs. N-terminally elongated domains in innexins may act to plug or manipulate hemichannel closure and provide a mechanism connecting the effect of hemichannel closure directly to apoptotic signaling transduction (Chen et al. 2016).

Inx2 of Spodoptera litura (Asian cotton leafworm)

Innexin 2, Inx2; Prp33, of 367 aas and 4 TMSs. It is a structural components of gap junctions, and is involved in gap junctional communication between germline and somatic cells which is essential for normal oogenesis (Bohrmann and Zimmermann 2008). In embryonic epidermis, it is required for epithelial morphogenesis as well as for keyhole formation during early stages of proventriculus development in response to wg signaling (Bauer et al. 2004). In follicle cells, it promotes the formation of egg chambers, in part through regulation of shg and baz at the boundary between germ cells and follicle cells. In inner germarial sheath cells, it is required for survival of early germ cells and for cyst formation (Mukai et al. 2011). 

Inx2 or Anon-37B-2 of Drosophila melanogaster (Fruit fly)

Innexin-2 of 358 aas and 6 TMSs. Innexin-2 can participate in many physiological processes during the development of R. americana (Neves et al. 2021).

Innexin-2 of Rhynchosciara americana

Invertebrate innexin, UNC-7

Invertebrates

UNC-7 of Caenorhabditis elegans

Innexin-14 (Protein Opu-14)

Inx-14 of Caenorhabditis elegans

Innexin-6 protein, Inx-6 or Opu-6, of 389 aas and 4 TMSs. A single INX-6 gap junction channel consists of 16 subunits, a hexadecamer, in contrast to chordate connexin channels, which consist of 12 subunits. The channel pore diameters at the cytoplasmic entrance and extracellular gap region are larger than those of connexin26 (Oshima et al. 2016). Nevertheless, the arrangements of the transmembrane helices and extracellular loops of the INX-6 monomer are highly similar to those of connexin-26 (Cx26). The INX-6 gap junction channel comprises hexadecameric subunits but reveals an N-terminal pore funnel consistent with Cx26. The helix-rich cytoplasmic loop and C-terminus are intercalated through an octameric hemichannel, forming a dome-like entrance that interacts with N-terminal loops in the pore (Oshima et al. 2016).

Worm

Inx-6 of Caenorhabditis elegans

Innexin Inx4 (Innexin-4) (Protein zero population growth)

Zpg of Drosophila melanogaster

Leech innexin, Inx6 (Kandarian et al. 2012; Firme et al. 2012)

Animals

Inx6 of Hirudo verbana

Pannexin-1 (PANX1) has been reported to form functional, single membrane, cell surface channels (Penuela et al., 2007). Pannexin1 is part of the pore forming unit of the P2X(7) receptor death complex (Locovei et al., 2007). It can catalyze ATP release from cells (Huang and Roper, 2010) and promote ATP signalling in mice (Suadicani et al. 2012). It also promotes acetaminophen liver toxicity by allowing it to enter the cell (Maes et al. 2016).  Pannexin1 and pannexin2 channels show quaternary similarities to connexons but different oligomerization numbers (Ambrosi et al., 2010). Pannexin 1 constitutes the large conductance cation channel of cardiac myocytes (Kienitz et al., 2011). Pannexin 1 (Px1, Panx1) and pannexin 2 (Px2, Panx2) underlie channel function in neurons and contribute to ischemic brain damage (Bargiotas et al., 2011). Single cysteines in the extracellular and transmembrane regions modulate pannexin 1 channel function (Bunse et al., 2011).  Spreading depression triggers migraine headaches by activating neuronal pannexin1 (panx1) channels  (Karatas et al. 2013).  The channel in the mouse orthologue opens upon apoptosis (Spagnol et al. 2014). Transports ATP out of the cell since L-carbenoxolone (a Panx1 channel blocker) inhibits ATP release from the nasal mucosa, but flufenamic acid (a connexin channel blocker) and gadolinium (a stretch-activated channel blocker) do not (Ohbuchi et al. 2014). CALHM1 (TC#1.N.1.1.1) and PANX1 both play roles in ATP release and downstream ciliary beat frequency modulation following a mechanical stimulus in airway epithelial cells (Workman et al. 2017). Pannexin1 may play a role in the pathogenesis of liver disease (Willebrords et al. 2018). Inhibition of pannexin1 channel opening may provide a novel approach for the treatment of drug (acetaminophen-induced)-induced hepatotoxicity (Maes et al. 2017). Pannexin-1 is necessary for capillary tube formation on Matrigel and for VEGF-C-induced invasion. It is highly expressed in HDLECs and is required for in vitro lymphangiogenesis (Boucher et al. 2018). cryo-EM structure of a pannexin 1 reveals unique motifs for ion selection and inhibition. The cryo-EM structure of a pannexin 1 revealed unique motifs for ion selection and inhibition (Michalski et al. 2020). In another study, Deng et al. 2020 obtained near-atomic-resolution structures of human and frog PANX1 determined by cryo-EM that revealed a heptameric channel architecture. Compatible with ATP permeation, the transmembrane pore and cytoplasmic vestibule were exceptionally wide. An extracellular tryptophan ring located at the outer pore created a constriction site, potentially functioning as a molecular sieve that restricts the sizes of permeable substrates. Pannexin 1 channels in renin-expressing cells influence renin secretion and homeostasis (DeLalio et al. 2020). Structures of human pannexin 1 have revealed ion pathways and mechanism of gating (Ruan et al. 2020). PANX1 is critical for functions such as blood pressure regulation, apoptotic cell clearance and human oocyte development. Ruan et al. 2020 presented several structures of human PANX1 in a heptameric assembly at resolutions of up to 2.8 Å, including an apo state, a caspase-7-cleaved state and a carbenoxolone-bound state. A gating mechanism was revealed that involves two ion-conducting pathways. Under normal cellular conditions, the intracellular entry of the wide main pore is physically plugged by the C-terminal tail. Small anions are conducted through narrow tunnels in the intracellular domain. These tunnels connect to the main pore and are gated by a long linker between the N-terminal helix and the first transmembrane helix. During apoptosis, the C-terminal tail is cleaved by caspase, allowing the release of ATP through the main pore. A carbenoxolone (a channel blocker)-binding site is embraced by W74 in the extracellular entrance. A gap-junction-like structure was observed as expected (Yen and Saier 2007; Chou et al. 2017). Navis et al. 2020 provided a review of the literature on Panx1 structural biology and known pharmacological agents that target it. The R217H mutation perturbs the conformational flexibility of the C-terminus, leading to channel dysfunction (Purohit and Bera 2021). Panx1 plays decisive roles in multiple physiological and pathological settings, including oxygen delivery to tissues, mucociliary clearance in airways, sepsis, neuropathic pain, and epilepsy. It exerts some of these roles in the context of purinergic signaling by providing a transmembrane pathway for ATP, but Panx1 can also act as a highly selective membrane channel for chloride ions without ATP permeability (Mim et al. 2021). Pannexin 1 regulates skeletal muscle regeneration by promoting bleb-based myoblast migration and fusion through a lipid based signaling mechanism (Suarez-Berumen et al. 2021).  Pannexin-1 activation by phosphorylation is crucial for platelet aggregation and thrombus formation (Metz and Elvers 2022). Data suggest that in response to hypotonic stress, the intact rat lens is capable of releasing ATP. This seems to be mediated via the opening of pannexin channels in a specific zone of the outer cortex of the lens (Suzuki-Kerr et al. 2022). Expression of pannexin1 in lung cancer brain metastasis and immune microenvironment has been reported (Abdo et al. 2023). Pannexin-1 (Panx1) hemichannels are non-selective transmembrane channels that play roles in intercellular signaling by allowing the permeation of ions and metabolites, such as ATP. Evidence suggests that Panx1 hemichannels control excitatory synaptic transmission. García-Rojas et al. 2023 studied the contribution of Panx1 to the GABAergic synaptic efficacy onto CA1 pyramidal neurons (PyNs) by using patch-clamp recordings and pharmacological approaches in wild-type and Panx1 knock-out (Panx1-KO) mice. Blockage of the Panx1 hemichannel with the mimetic peptide increased the synaptic level of endocannabinoids (eCB) and the activation of cannabinoid receptors type 1 (CB1Rs), which resulted in a decrease in hippocampal GABAergic efficacy, shifting excitation/inhibition (E/I) balance toward excitation and facilitating the induction of long-term potentiation. Thus, Panx1 strongly influences neuronal excitability and plays a key role in shaping synaptic changes affecting the amplitude and direction of plasticity as well as learning and memory processes (García-Rojas et al. 2023). Genetic deletion of PANX1 mitigates kidney tubular cell death, oxidative stress and mitochondrial damage after renal ischemia/reperfusion (I/R) injury through enhanced mitophagy. Mechanistically, PANX1 disrupts mitophagy by influencing the ATP-P2Y-mTOR signal pathway. Thus, PANX1 could be a biomarker for acute kidney injury (AKI) and a therapeutic target to alleviate AKI caused by I/R injury (Su et al. 2023). Blocking pannexin 1 channels alleviates peripheral inflammatory pain but not paclitaxel-induced neuropathy (Lemes et al. 2024).  Cx43 hemichannels and panx1 channels contribute to ethanol-induced astrocyte dysfunction and damage (Gómez et al. 2024).  Pannexin1 mediates early-life seizure-induced social behavior deficits (Obot et al. 2024). The small molecule raptinal can simultaneously induce apoptosis and inhibit PANX1 activity (Santavanond et al. 2024).  A heterozygous missense variant of PANX1 causes human oocyte death and female infertility (Zhou et al. 2024).

Vertebrates

Pannexin-1 of Homo sapiens

Pannexin1 and pannexin2 (pannexin-2; pannexin 2) channels show quaternary similarities to connexons but different oligomerization numbers (Ambrosi et al., 2010). Pannexin 1 (Px1, Panx1) and pannexin 2 (Px2, Panx2) underlie channel function in neurons and contribute to ischemic brain damage (Bargiotas et al., 2011). PANX2 channels participate in multiple physiological processes including skin homeostasis, neuronal development, and ischemia-induced brain injury. He et al. 2023 presented a cryo-EM structure of human PANX2, which revealed pore properties contrasting with those of the intensely studied paralog, PANX1. The extracellular selectivity filter, defined by a ring of basic residues, more closely resembles that of the distantly related volume-regulated anion channel (VRAC) LRRC8A (TC# 1.A.25.3.1), rather than PANX1. Furthermore, PANX2 displays a similar anion permeability sequence as VRAC, and PANX2 channel activity is inhibited by a commonly used VRAC inhibitor, DCPIB. The shared channel properties between PANX2 and VRAC may complicate dissection of their cellular functions through pharmacological manipulation (He et al. 2023). The cryo-EM structure of the human heptameric PANX2 channel has been solved (Zhang et al. 2023). It is a large-pore ATP-permeable channel with critical roles in various physiological processes, such as the inflammatory response, energy production and apoptosis. Its dysfunction is related to numerous pathological conditions including ischemic brain injury, glioma and glioblastoma multiforme. The structure was solved at a resolution of 3.4 Å. The Panx2 structure assembles as a heptamer, forming an exceptionally wide channel pore across the transmembrane and intracellular domains, compatible with ATP permeation. Comparing Panx2 with Panx1 structures in different states reveals that the Panx2 structure corresponds to an open channel state. A ring of seven arginine residues located at the extracellular entrance forms the narrowest site of the channel, which serves as the critical molecular filter controlling the permeation of substrate molecules. This was further verified by molecular dynamics simulations and ATP release assays. These studies revealed the architecture of the Panx2 channel and provided insights into the molecular mechanism of its channel gating (Zhang et al. 2023).

Vertebrates

Pannexin-2 of Homo sapiens (Q96RD6)

Pannexin-3, PANX3, of 392 aas and 5 TMSs, is reported to form functional, single membrane, cell surface channels (Penuela et al., 2007)). It functions as an ER Ca²⁺ channel, hemichannel, and gap junction to promote osteoblast differentiation (Ishikawa et al., 2011). However, Panx3 expression in osteoblasts is not required for postnatal bone remodeling (Yorgan et al. 2019).

Vertebrates

Pannexin-3 of Homo sapiens (gi16418453)

Pannexin 1a, Panx1a, of 417 aas and 4 TMSs, an ATP channel.  Aromatic-aromatic interaction involving Trp123 and Tyr205 in TMSs 2 and 3, respectively are important for the assembly and trafficking of the Zebrafish Panx1a membrane channel (Timonina et al. 2020).

Panx1a of Danio rerio (Zebrafish) (Brachydanio rerio)

Mouse Pannexin 1 of 426 aas and 4 TMSs.  Pannexins are ubiquitously expressed in human and mouse tissues. Pannexin 1 (Panx1), the most thoroughly characterized member of this family, forms plasmalemmal membrane channels permeable to relatively large molecules, such as ATP. Although human and mouse Panx1 amino acid sequences are conserved in the presently known regulatory sites involved in trafficking and modulation of the channel, differences occur in the N- and C-termini of the protein.  Cibelli et al. 2023 used a neuroblastoma cell line to study the activation properties of endogenous mPanx1 and exogenously expressed hPanx1. Dye uptake and electrophysiological recordings revealed that in contrast to mouse Panx1, the human ortholog is insensitive to stimulation with high extracellular K⁺ but responds similarly to activation of the purinergic P2X7 receptor. The two most frequent Panx1 polymorphisms found in the human population, Q5H (rs1138800) and E390D (rs74549886), exogenously expressed in Panx1-null N2a cells revealed that regarding P2X7 receptor mediated Panx1 activation, the Q5H mutant is a gain of function whereas the E390D mutant is a loss of function variant. Collectively, they demonstrated differences in the activation between human and mouse Panx1 orthologs and suggest that these differences may have translational implications for studies where Panx1 has been shown to have a significant impact (Cibelli et al. 2023).

Pannexin 1 of Mus musculus

The volume-regulated Anion Channel, VRAC, or volume-sensitive outward rectifying anion channel, VSOR. It is also called the SWELL1 protein. It consists of the leucine-rich repeat-containing protein 8A, with an N-terminal pannexin-like domain, LRRC8A, together with other LRRC8 subunits (B, C, D and E). The first two TMSs of the 4 TMS LRRC8 proteins appear as DUF3733 in CDD (Abascal and Zardoya, 2012). The C-terminal soluble domain shows sequence similarity to the heme-binding protein, Shv, and pollen-specific leucine-rich repeat extension-like proteins (3.A.20.1.1).  The volume-regulated anion channel, VRAC, has LRRC8A as a VRAC component. It forms heteromers with other LRRC8 membrane proteins (Voss et al. 2014). Genomic disruption of LRRC8A ablated VRAC currents. Cells with disruption of all five LRRC8 genes required LRRC8A cotransfection with other LRRC8 isoforms to reconstitute VRAC currents. The isoform combination determined the VRAC inactivation kinetics. Taurine flux and regulatory volume decrease also depended on LRRC8 proteins. Thus, VRAC defines a class of anion channels, suggesting that VRAC is identical to the volume-sensitive organic osmolyte/anion channel VSOAC, and explains the heterogeneity of native VRAC currents (Voss et al. 2014).  Point mutations in two amino-acyl residues (Lys98 and Asp100 in LRRC8A and equivalent residues in LRRC8C and -E) upon charge reversal, alter the kinetics and voltage-dependence of inactivation (Ullrich et al. 2016). Using cryo-electron microscopy and X-ray crystallography, Deneka et al. 2018 and Kasuya et al. 2018  determined the structures of a homomeric channel of the obligatory subunit LRRC8A. This protein conducts ions and has properties in common with endogenous heteromeric channels. Its modular structure consists of a transmembrane pore domain followed by a cytoplasmic leucine-rich repeat domain. The transmembrane domain, which is structurally related to connexins, is wide towards the cytoplasm but constricted on the outside by a structural unit that acts as a selectivity filter. An excess of basic residues in the filter and throughout the pore attracts anions by electrostatic interaction (Deneka et al. 2018). The structure shows a hexameric assembly, and the transmembrane region features a topology similar to gap junction channels. The LRR region, with 15 leucine-rich repeats, forms a long, twisted arc. The channel pore is located along the central axis and constricted on the extracellular side, where highly conserved polar and charged residues at the tip of the extracellular helix contribute to the permeability to anions and other osmolytes. Two structural populations were identified, corresponding to compact and relaxed conformations. Comparing the two conformations suggests that the LRR region is flexible and mobile with rigid-body motions, which might be implicated in structural transitions on pore opening (Kasuya et al. 2018). VRAC is inhibited by Tamoxifen and Mefloquine (Lee et al. 2017). The intracellular loop connecting TMSs 2 and 3 of LRRC8A and the first extracellular loop connecting transmembrane domains 1 and 2 of LRRC8C, LRRC8D, or LRRC8E are essential for VRAC activity (Yamada and Strange 2018). The N termini of the LRRC8 subunits may line the cytoplasmic portion of the VRAC pore, possibly by folding back into the ion permeation pathway (Zhou et al. 2018).  On the adipocyte plasma membrane, the SWELL1-/LRRC8 channel complex activates in response to increases in adipocyte volume in the context of obesity. SWELL1 is required for insulin-PI3K-AKT2 signalling to regulate adipocyte growth and systemic glycaemia (Gunasekar et al. 2019).  Activation of Swell1 in microglia suppresses neuroinflammation and reduces brain damage in ischemic stroke (Chen et al. 2023).

Animals

The VRAC channel consisting of LRRC8A together with one or two of the subunits, LRRC8B, LRRC8C, LRRC8D and/or LRRC8E of Homo sapiens (Q8IWT6)

The LRRC8B homologue of 480 aas.  Its cytoplasmic domains are regulators of channel activity by allosteric mechanisms (Deneka et al. 2021).

Animals

LRRC8B of Ciona intestinalis (Transparent sea squirt) (Ascidia intestinalis)

Uncharacterized protein of 467 aas

Animals

UP of Branchiostoma floridae (Florida lancelet) (Amphioxus)

Uncharacterized ADP-binding protein of 1311 aas and 2 TMSs.  May be involved in defense responses.

Plants

UP of Oryza sativa

Volume-regulated anion channel subunit LRRC8B-like protein of 666 aas and 4 TMSs.

LRRC8B of Mizuhopecten yessoensis

Uncharacterized protein of 610 aas and 4 TMSs.  It is of the Pannexin-like Superfamily.

UP of Thelohanellus kitauei

Leucine-rich repeat-containing protein 59, LRRC59, of 307 aas and 1 C-terminal TMS. It is a tail-anchored protein  that localizes to the ER and the nuclear envelope and is required for nuclear import of FGF1. It might regulate nuclear import by facilitating interaction with the nuclear import machinery and by transporting cytosolic FGF1 to, and possibly through, the nuclear pore (TC# 1.I.1) (Zhen et al. 2012). LRRC59 is post-translationally inserted into ER-derived membranes, possibly by diffusion (Blenski and Kehlenbach 2019).

LRRC59 of Homo sapiens

Sr35 of 919 aas with possibly 3 TMSs, one N-terminal, one at about residue 380 and one near the C-terminus of the protein (Förderer et al. 2022).

Sr35 of Triticum monococcum

Zar1 resistosome of 852 aas and possibly about 3 TMSs of low hydrophobicity, is a calcium-permeable channel that triggers plant immune signalling (Bi et al. 2021). It forms a pentameric channel. It is a nucleotide-binding leucine-rich repeat receptor (NLR protein). Homologues in animals are called inflammasomes (Davis et al. 2011).

Zar1 of Arabidopsis thaliana