1.A.17 The Calcium-dependent Chloride Channel (Ca-ClC) Family
The Anoctamin Superfamily of cation and anion channels, as well as lipid scramblases, includes three functionally characterized families: the Anoctamin (ANO), Transmembrane Channel (TMC) and Ca2+-permeable Stress-gated Cation Channel (CSC) families. There are also four families of functionally uncharacterized proteins, which are referred to as the Anoctamin-like (ANO-L), Transmembrane Channel-like (TMC-L), and CSC-like (CSC-L1 and CSC-L2) families (Medrano-Soto et al. 2018). Protein clusters and trees showing the relative relationships among the seven families were constructued, and topological analyses suggested that the members of these families have essentially the same topologies. Comparative examination of these homologous families provided insight into possible mechanisms of action, indicated the currently recognized organismal distributions of these proteins, and suggested drug design potential for the disease-related channel proteins (Medrano-Soto et al. 2018). During the first postnatal week of mouse development, the current amplitude grew, and transducer adaptation became faster and more effective, due partly to a developmental switch from TMC2- to TMC1-containing channels and partly to an increase in channel expression (Goldring et al. 2019). Nist-Lund et al. 2019 designed TMC1 and TMC2 gene replacement therapies which corrected hearing and vertigo disorders. TMC1 and TMC2 are hair cell transduction channels (Jia et al. 2019). Signaling through the interleukin-4 and interleukin-13 receptor complexes regulates cholangiocyte TMEM16A expression and biliary secretion (Dutta et al. 2020). ANOs 3-7 in the anoctamin/Tmem16 family are intracellular membrane proteins (Duran et al. 2012). Both anion channels (such as TMEM16A) and phospholipid scramblases (such as TMEM16F) are activated by intracellular Ca2+ in the low microM range, but many divalent cations at mM concentrations further activate (Nguyen et al. 2021).
Impaired chloride transport can cause diseases as diverse as cystic fibrosis, myotonia, epilepsy, hyperekplexia, lysosomal storage disease, deafness, renal salt loss, kidney stones and osteopetrosis. These disorders are caused by mutations in genes belonging to non-related gene families, including CLC chloride channels and GABA- and glycine-receptors. Diseases due to mutations in Anoctamin 1 TMEM16E and bestrophin 1 might be due to a loss of Ca2+-activated Cl- channels, although this remains to be shown (Planells-Cases and Jentsch, 2009). The evolution and functional divergence of anoctamin family members has been reported (Milenkovic et al. 2010). Some, but not all TMEM16 homologues can catalyze phospholipid flipping as phospholipid scramblases in addition to their roles as ion channels (Malvezzi et al. 2013). Compromised anoctamin function causes a wide range of diseases, such as hearing loss (ANO2), bleeding disorders (ANO6), ataxia and dystonia (ANO3), persistent borrelia and mycobacteria infection (ANO10), skeletal syndromes like gnathodiaphyseal dysplasia and limb girdle muscle dystrophy (ANO5), and cancer (ANO1) (Kunzelmann et al. 2015). Calcium-activated chloride channels (CaCCs) in response to calcium release from intracellular stores, mediated by G-protein coupled receptors, can lead to CaCC activation, and prominent inflammatory mediators like bradykinin or serotonin also stimulate CaCCs via such a mechanism (Salzer and Boehm 2019). The transport of bicarbonate (HCO3-) by anion channels and its relevance to human diseases has been discussed (Shin et al. 2020).
Calcium-dependent chloride channels are required for normal electrolyte and fluid secretion, olfactory perception, and neuronal and smooth muscle excitability in animals (Pang et al. 2013). Treatment of bronchial epithelial cells with interleukin-4 (IL-4) causes increased calcium-dependent chloride channel activity. Caputo et al., 2008 performed a global gene expression analysis to identify membrane proteins that are regulated by IL-4. TMEM16A is associated with calcium-dependent chloride current, as measured with halide-sensitive fluorescent proteins, short-circuit current, and patch-clamp techniques. Their results indicated that TMEM16A is an intrinsic constituent (9 putative TMSs) of the calcium-dependent chloride channel. These results have been confirmed and extended by Yang et al., 2008 and Ferrera et al., 2009. Transmembrane protein 16B (TMEM16B) is also a Ca2+-activated Cl- channel but with different voltage dependence and unitary conductance (Galietta, 2009). Scudieri et al. (2011) reported that TMEM16A has a putative structure consisting of eight transmembrane domains with both the amino- and the carboxy-termini protruding in the cytosol. TMEM16A is also characterized by the existence of different protein variants generated by alternative splicing. TMEM16B (anoctamin-2) is also associated with CaCC activity although with different properties. TMEM16B-dependent channels require higher intracellular Ca2+ concentrations and have faster activation and deactivation kinetics. Expression of other anoctamins is instead devoid of detectable channel activity. These proteins, such as TMEM16F (anoctamin-6), may have different functions. Yue et al. 2019 have presented a comparative overview of the diverse functions of TMC channels in different species.
All vertebrate cells regulate their cell volume by activating chloride channels thereby activating regulatory volume decrease. Almaça et al., 2009 showed that the Ca2+-activated Cl- channel TMEM16A together with other TMEM16 proteins are activated by cell swelling through an autocrine mechanism that involves ATP release and binding to purinergic P2Y(2) receptors. TMEM16A channels are activated by ATP through an increase in intracellular Ca2+ and a Ca2+-independent mechanism engaging extracellular-regulated protein kinases (ERK1/2). The ability of epithelial cells to activate a Cl- conductance upon cell swelling, and to decrease their cell volume was dependent on TMEM16 proteins. Activation was reduced in the colonic epithelium and in salivary acinar cells from mice lacking expression of TMEM16A. Thus, TMEM16 proteins appear to be a crucial component of epithelial volume-regulated Cl- channels and may also have a function during proliferation and apoptotic cell death.
Interstitial cells of Cajal (ICC) generate pacemaker activity (slow waves) in gastrointestinal (GI) smooth muscles. Several conductances, such as Ca2+-activated Cl- channels (CaCC) and non-selective cation channels (NSCC) have been suggested to be involved in slow wave depolarization. Hwang et al., 2009 investigated the expression and function of anoctamin 1 (ANO1), encoded by Tmem16a, which is highly expressed in ICC. GI muscles express splice variants of the Tmem16a transcript in addition to other paralogues of the Tmem16a family. ANO1 protein is expressed abundantly and specifically in ICC in all regions of the murine, non-human primate (Macaca fascicularis) and human GI tracts. CaCC blocking drugs, niflumic acid and 4,4-diisothiocyano-2,2-stillbene-disulfonic acid (DIDS) reduced the frequency and blocked slow waves in murine, primate, human small intestine and stomach in a concentration-dependent manner. Slow waves failed to develop by birth in mice homozygous for a null allele of Tmem16a and did not develop subsequent to birth in organ culture, as in wildtype and heterozygous muscles. These data demonstrate the fundamental role of ANO1 in the generation of slow waves in GI ICC (Hwang et al., 2009).
The calcium-activated chloride channel anoctamin1 (ANO1; TMEM16A) is fundamental for the function of epithelial organs, and mice lacking ANO1 expression exhibit transport defects and a pathology similar to that of cystic fibrosis. They also show a general defect of epithelial electrolyte transport. Schreiber et al., (2010) analyzed expression of all ten members (ANO1-ANO10) in a broad range of murine tissues and detected predominant expression of ANO1, 6, 7, 8, 9, 10 in epithelial tissues, while ANO2, 3, 4, 5 are common in neuronal and muscle tissues. When expressed in Fisher Rat Thyroid (FTR) cells, all ANO proteins localized to the plasma membrane, but only ANO1, 2, 6, and 7 produced Ca2+-activated Cl- conductance. In contrast, ANO9 and ANO10 suppressed baseline Cl- conductance, and coexpression of ANO9 with ANO1 inhibited ANO1 activity. Patch clamping of ANO-expressing FRT cells indicated that apart from ANO1, ANO6 and 10 produced chloride currents, but with very different Ca2+ sensitivity and activation time. Thus, each tissue expresses a set of anoctamins that form cell- and tissue-specific Ca2+-dependent Cl- channels (Schreiber et al., 2010).
In all animal cells, phospholipids are asymmetrically distributed between the outer and inner leaflets of the plasma membrane. This asymmetrical phospholipid distribution is disrupted in various biological systems. For example, when blood platelets are activated, they expose phosphatidylserine (PtdSer) to trigger the clotting system. The PtdSer exposure is believed to be mediated by Ca2+-dependent phospholipid scramblases that transport phospholipids bidirectionally. Suzuki et al. (2010) showed that TMEM16F (transmembrane protein 16F) is essential for the Ca2+-dependent exposure of phosphatidylserine on the cell surface. Wild-type and mutant forms of TMEM16F were localized to the plasma membrane and conferred Ca2+-dependent scrambling of phospholipids. A patient with Scott syndrome, which results from a defect in phospholipid scrambling activity, was found to carry a mutation at a splice-acceptor site of the gene encoding TMEM16F, causing premature termination of the protein (Suzuki et al., 2010).
The Ca-ClC anoctamin (Tmem16) gene family was first identified by bioinformatic analysis in 2004. In 2008, it was shown independently by 3 laboratories that the first two members (Tmem16A and Tmem16B) of this 10-gene family are Ca2+-activated Cl- channels. Because these proteins are thought to have 8 transmembrane domains and be anion-selective channels, the alternative name, Anoctamin (anion and octa=8), has been proposed. It is not clear that all members of this family are anion channels or have the same 8-transmembrane domain topology. Between 2008 and 2011, there have been nearly 100 papers published on this gene family (Duran and Hartzell, 2011). Ano1 has been linked to cancer while mutations in Ano5 are linked to several forms of muscular dystrophy (LGMDL2 and MMD-3). Mutations in Ano10 are linked to autosomal recessive spinocerebellar ataxia, while mutations in Ano6 are linked to Scott syndrome, a rare bleeding disorder. Duran and Hartzell (2011) have reviewed the physiology and structure-function relationships of the Tmem16 gene family.
Tmc1 and Tmc2 (TC#s 1.A.17.4.6 and 1.A.17.4.1, respectively) may play a role in hearing and are required for normal function of cochlear hair cells, possibly as Ca2+ channels or Ca2+ channel subunits (Kim and Fettiplace 2013) (see also family 1.A.82). Mice lacking both channels lack hair cell mechanosensory potentials (Kawashima et al. 2011). There are 8 members of this family in humans, 1 in Drosophila and 2 in C. elegans. One of the latter two is expressed in mechanoreceptors (Smith et al. 2010). Tmc-1 is a sodium-sensitive cation Ca2+ channel required for salt (Na+) chemosensation in C. elegans where it is required for salt-evoked neuronal activity and behavioural avoidance of high concentrations of NaCl (Chatzigeorgiou et al. 2013). Most evidence is consistent with TMCs being pore-forming subunits of the hair-cell transduction channel (Corey and Holt 2016).
Hair cells express two molecularly and functionally distinct mechanotransduction channels with different subcellular distributions. One is activated by sound and is responsible for sensory transduction. This sensory transduction channel is expressed in hair cell stereocilia, and its activity is affected by mutations in the genes encoding the transmembrane proteins TMHS (TC# 1.A.82.1.1), TMIE (TC# 9.A.30.1.1), TMC1 and TMC2 (family 1.A.17.4) (Wu et al. 2016). The other is the Piezo2 channel (TC# 1.A.75.1.2).
Mutations in transmembrane channel-like gene 1 (TMC1/Tmc1) cause dominant or recessive hearing loss in humans and mice. Tmc1 mRNA is specifically expressed in neurosensory hair cells of the inner ear. Cochlear neurosensory hair cells of Tmc1 mutant mice fail to mature into fully functional sensory receptors and exhibit concomitant structural degeneration that could be a cause or an effect of the maturational defect. The molecular and cellular functions of TMC1 protein are substantially unknown due, at least in part, to in situ expression levels that are prohibitively low for direct biochemical analysis (Labay et al., 2010). TMCs are biomarkers for prognosis and immunotherapeutic response, which can pave the way for further investigation of the tumor-infiltrating mechanisms and therapeutic potentials of TMCs in cancer (Song et al. 2021).
There are seven additional mammalian TMC paralogs. An initial PSORT-II analysis of human and mouse TMC proteins did not detect N-terminal signal sequences or other trafficking signals. The TMC proteins are predicted to contain 6-10 TMSs and a novel, conserved region termed the TMC domain. Human TMC6 (also known as EVER1) and TMC8 (EVER2) proteins are retained in the endoplasmic reticulum (Labay et al., 2010). Truncating mutations of EVER1 and EVER2 cause epidermodysplasia verruciformis (EV; MIM 226400), characterized by susceptibility to cutaneous human papilloma virus infections and associated non-melanoma skin cancers. Sound stimuli elicit movement of the stereocilia that make up the hair bundle of cochlear hair cells, putting tension on the tip links connecting the stereocilia and thereby opening mechanotransducer (MT) channels. Tmc1 and Tmc2, two members of the transmembrane channel-like family, are necessary for mechanotransduction. Kim et al. (2013) recorded MT currents elicited by hair bundle deflections in mice with null mutations of Tmc1, Tmc2, or both. During the first postnatal week. They observed normal MT currents in hair cells lacking Tmc1 or Tmc2; however, in the absence of both isoforms, we recorded a large MT current that was phase-shifted 180°, being evoked by displacements of the hair bundle away from its tallest edge rather than toward it as in wild-type hair cells. The anomalous MT current in hair cells lacking Tmc1 and Tmc2 was blocked by FM1-43, dihydrostreptomycin, and extracellular Ca2+ at concentrations similar to those that blocked wild type. MT channels in the double knockouts carried Ca2+ with a lower permeability than wild-type or single mutants. The MT current in double knockouts persisted during exposure to submicromolar Ca2+, even though this treatment destroyed the tip links. Kim et al. (2013) concluded that the Tmc isoforms do not themselves constitute the MT channel but are essential for targeting and interaction with the tip link. Changes in the MT conductance and Ca2+ permeability observed in the absence of Tmc1 mutants may stem from loss of interaction with protein partners in the transduction complex. See also (Kim et al. 2013).
Ion channels promote the development and progression of tumors. TMEM16A is overexpressed in several tumor types. The role of TMEM16A in gliomas and the potential underlying mechanisms were analyzed by Liu et al. 2014. TMEM16A was abundant in various grades of gliomas and cultured glioma cells. Knockdown of TMEM16A suppressed cell proliferation, migration and invasion. Nuclear factor kappaB (NFkappaB) was activated by overexpression of TMEM16A, and, TMEM16A regulated the expression of NFkappaB-mediated genes, including cyclin D1, cyclin E and cmyc, involved in cell proliferation, and matrix metalloproteinases (MMPs)2 and MMP9, which are associated with the migration and invasion of glioma cells.
Activation of the TMEM16A-encoded CaCC (ANO1) is mediated by Ca2+, Sr2+, and Ba2+. Mg2+ competes with Ca2+ in binding to the divalent-cation binding site without activating the channel. The anion occupancy in the pore-as revealed by the permeability ratios of these anions appeared to be inversely correlated with the apparent affinity of the ANO1 inhibition by niflumic acid (NFA) (Ni et al. 2014). On the other hand, NFA inhibition was neither affected by the degree of the channel activation nor influenced by the types of divalent cations used for channel activation. These results suggest that the NFA inhibition of ANO1 is likely mediated by altering pore function, not through changing channel gating.
Ca2+-activated Cl- channels (CaCCs) are a class of Cl- channels activated by intracellular Ca2+ that are known to mediate numerous physiological functions. In 2008, the molecular identity of CaCCs was found to be anoctamin 1 (ANO1/TMEM16A). Its roles have been studied in electrophysiological, histological, and genetic aspects. ANO1 is known to mediate Cl- secretion in secretory epithelia such as airways, salivary glands, intestines, renal tubules, and sweat glands (Oh and Jung 2016). ANO1 is a heat sensor activated by noxious heat in somatosensory neurons and mediates acute pain sensation as well as chronic pain. ANO1 is also observed in vascular as well as airway smooth muscles, controlling vascular tone as well as airway hypersensitivity. ANO1 is upregulated in numerous types of cancers and thus thought to be involved in tumorigenesis. ANO1 is also found in proliferating cells. In addition to ANO1, involvement of its paralogs in pathophysiological conditions has also been reported. ANO2 is involved in olfaction, whereas ANO6 works as a scramblase whose mutation causes a rare bleeding disorder, the Scott syndrome. ANO5 is associated with muscle and bone diseases (Oh and Jung 2016). An X-ray crystal structure of a fungal TMEM16 has been reported, which explains a precise molecular gating mechanism as well as ion conduction or phospholipid transport across the plasma membrane (Brunner et al. 2014).
Polar and charged lipid headgroups are believed to move through the low-dielectric environment of the membrane by traversing a hydrophilic groove on the membrane-spanning surface of the protein. Bethel and Grabe 2016 explored the membrane-protein interactions involved in lipid scrambling. A global pattern of charged and hydrophobic surface residues bends the membrane in a large-amplitude sinusoidal wave, resulting in bilayer thinning across the hydrophilic groove. Atomic simulations uncovered two lipid headgroup- interaction sites flanking the groove. The cytoplasmic site nucleates headgroup-dipole stacking interactions that form a chain of lipid molecules that penetrate into the groove. In two instances, a cytoplasmic lipid interdigitates into this chain, crosses the bilayer, and enters the extracellular leaflet, and the reverse process happens twice as well. Several family members appear to all bend the membrane - even those that lack scramblase activity. Sequence alignments show that the lipid interaction sites are conserved in many family members but less so in those with reduced scrambling ability (Bethel and Grabe 2016).
TMEM16A forms a dimer with two pores. Dang et al. 2017 presened de novo atomic structures of the transmembrane domains of mouse TMEM16A in nanodiscs and in lauryl maltose neopentyl glycol as determined by single-particle electron cryo-microscopy. These structures reveal the ion permeation pore and represent different functional states (Dang et al. 2017). The structure in lauryl maltose neopentyl glycol has one Ca2+ ion resolved within each monomer with a constricted pore; this is likely to correspond to a closed state, because a CaCC with a single Ca2+ occupancy requires membrane depolarization in order to open. The structure in nanodiscs has two Ca2+ ions per monomer, and its pore is in a closed conformation. Ten residues are distributed along the pore that interact with permeant anions and affect anion selectivity, and seven pore-lining residues cluster near pore constrictions and regulate channel gating (Dang et al. 2017).
Overexpression of TMEM16A may be associated with cancer progression. Zhang et al. 2017 showed that four flavinoids - luteolin, galangin, quercetin and fisetin - have inhibitory IC50 values ranging from 4.5 to 15 muM. These flavonoids inhibited TMEM16A currents as well as cell proliferation and migration of LA795 cancer cells. A good correlation between TMEM16A current inhibition and cell proliferation and migration was observed (Zhang et al. 2017).
Similar to TMEM16F and 16E, seven TMEM16 family members were found to carry a domain (SCRD; scrambling domain) spanning the fourth and fifth TMSs that conferred scrambling ability to TMEM16A. By introducing point mutations into TMEM16F, Gyobu et al. 2017 found that a lysine in the fourth TMS of the SCRD as well as an arginine in the third and a glutamic acid in the sixth transmembrane segment were important for exposing phosphatidylserine from the inner to the outer leaflet. These results suggest that TMEM16 provides a cleft containing hydrophilic 'stepping stones' for the outward translocation of phospholipids (Gyobu et al. 2017).
Hair cells in the inner ear convert mechanical stimuli provided by sound waves and head movements into electrical signals. Several mechanically evoked ionic currents with different properties have been recorded in hair cells. In 2018, searches for the protein(s) that form the underlying ion channel(s) were not definitive. The mechanoelectrical transduction (MET) channel is near the tips of stereocilia in hair cell. It is responsible for sensory transduction (Qiu and Müller 2018). Several components of the sensory mechanotransduction machinery have been identified, including the multi-transmembrane proteins tetraspan membrane protein in hair cell stereocilia (TMHS)/LHFPL5, transmembrane inner ear (TMIE) and transmembrane channel-like proteins 1 and 2 (TMC1/2). However, there remains considerable uncertainty regarding the molecules that form the channel pore. In addition to the sensory MET channel, hair cells express the mechanically gated ion channel PIEZO2, which is localized near the base of stereocilia and is not essential for sensory transduction. The function of PIEZO2 in hair cells is not entirely clear, but it may play a role in damage sensing and repair processes. Additional stretch-activated channels of unknown molecular identity are found to localize at the basolateral membrane of hair cells. Cunningham and Müller 2018 reviewed knowledge regarding the different mechanically gated ion channels in hair cells and discuss open questions concerning their molecular compositions and functions.
Microdomains formed by proteins of the endoplasmic reticulum and plasma membrane play a key role in store-operated Ca2+ entry (SOCE). Ca2+ release through inositol 1,4,5-trisphosphate receptor (IP3R) and subsequent Ca2+ store depletion activate STIM (stromal interaction molecules) proteins, sensors of intraluminal Ca2+, which, in turn, open the Orai channels in the plasma membrane. Downstream to this process, TRPC (transient receptor potential-canonical) calcium permeable channels could be activated. Kolesnikov et al. 2021 found that local Ca2+ entry through TRPC1 channels activated endogenous Ca2+-activated chloride channels (CaCCs) with properties similar to Anoctamin6 (TMEM16F). Their outward rectification is dependent on the membrane potential (Kolesnikov et al. 2021).
TMEM16F is an enigmatic Ca2+-activated phospholipid scramblase (CaPLSase) that passively transports phospholipids down their chemical gradients and mediates blood coagulation, bone development and viral infection. Le et al. 2019 identified an inner activation gate, formed of three hydrophobic residues, F518, Y563 and I612, in the middle of the phospholipid permeation pathway. Disrupting the inner gate alters phospholipid permeation. Lysine substitutions of F518 and Y563 lead to constitutively active CaPLSases that bypass Ca2+-dependent activation. Strikingly, an analogous lysine mutation to TMEM16F-F518 in TMEM16A (L543K) is sufficient to confer CaPLSase activity to this Ca2+-activated Cl- channel (Le et al. 2019).
Both lipid and ion translocation by Ca2+-regulated TMEM16 transmembrane proteins utilizes a membrane-exposed hydrophilic groove, several conformations of which are observed in TMEM16 protein structures. From analyses of atomistic molecular dynamics simulations of Ca2+-bound nhTMEM16, the mechanism of a conformational transition of the groove from membrane-exposed to occluded involves the repositioning of TMS4 following its disengagement from a TMS3/TMS4 interaction interface (Khelashvili et al. 2019). Residue L302 is a key element in the hydrophobic TMS3/TMS4 interaction patch that braces the open-groove conformation, which should be changed by an L302A mutation. The structure of the L302A mutant determined by cryo-EM revealed a partially closed groove that could translocate ions but not lipids. This was corroborated with functional assays showing severely impaired lipid scrambling, but robust channel activity by L302A (Khelashvili et al. 2019). Membrane lipids are both the substrates and a mechanistically responsive environment for TMEM16 scramblase proteins (Khelashvili et al. 2019).
Scramblases, have properties that facilitate lipid flip-flop from one membrane leaflet to another. Scramblases and similar transmembrane proteins could also affect the translocation of other amphiphilic molecules, including cell-penetrating (CP) or antimicrobial peptides (AMPs). Bartoš et al. 2021 studied the effect of transmembrane proteins on the translocation of amphiphilic peptides through the membrane. They demonstrate that transmembrane proteins with a hydrophilic patch enhance the translocation of amphiphilic peptides by stabilizing the peptide in the membrane.
The last 4 TMSs in members of TC subfamily 1.A.17.5 show sequence similarity to a family of 5 TMS proteins in TC family 9.B.306. Thus, the latter may have been the precursor of the calcium-recognition domain of the anoctamins (see description of TC family 9.B.306).
The reactions believed to be catalyzed by channels of the Ca-ClC family, in addition to lipid scrambling, are:
Cl- (out) ⇌ Cl- (in)
Cations (e.g., Ca2+) (out) ⇌ Cations (e.g., Ca2+) (in)
The plasma membrane Ca2 -activated chloride (IClCa) channel, TMEM16A (Anoctamin 1a; ANO1a) (Huang et al., 2012; Chen et al. 2011). The mouse orthologue (Q8BHY3), TMEM16A (956aas), is localized to the apical membranes of epithelia as well as intracellular membranes in many cell types. Knockout mice show diminished rhythmic contraction of gastric smooth muscle (Huang et al., 2009). ANO1 is also required for normal tracheal development (Ousingsawat et al., 2009). Expression is upregulated by epidermal growth factor (Mroz and Keely, 2012). Novel 5-substituted benzyloxy-2-arylbenzofuran-3-carboxylic acids are inhibitors (Kumar et al., 2012). TMEM16A channels contribute to the myogenic response in cerebral arteries (Bulley et al., 2012). Membrane stretch activates arterial myocyte TMEM16A channels, leading to membrane depolarization and vasoconstriction. A local Ca2+ signal generated by nonselective cation channels stimulates TMEM16A channels to induce myogenic constriction (Bulley et al., 2012). Ca2+/calmodulin activates bicarbonate (anion) transport (Jung et al. 2012). The protein exists in the membrane as a homodimer where the cytoplasmic N-terminus functions in dimerization (Tien et al. 2013). TMSs 5-6 of the 8 TMSs may comprise parts of the pore-loop that controls Cl- conductance (Adomaviciene et al. 2013). ANO1 confers IClCa in retinal neurons and acts as an intrinsic regulator of the presynaptic membrane potential during synaptic transmission (Jeon et al. 2013). TMEM16A may be a primary driver of the "Grow" (tumor proliferation) or "Go"(metastasis) model for cancer progression, in which TMEM16A expression acts to balance tumor proliferation and metastasis via its promoter methylation (Shiwarski et al. 2014). Regulation of TMEM16A/16B by Ca2+ is mediated by preassociated apo-calmodulin (Yang et al. 2014) as well as CaMKIIδ (Gui et al. 2015). Because the Cl- channel is the only active ion-selective conductance with a reversal potential that lies within the dynamic range of spiral ganglion neurons (SGN) action potentials, developmental alteration of [Cl-], and hence the equilibrium potential for Cl- (ECl), transforms the pre- to the post-hearing phenotype (Zhang et al. 2015). Four basic residues involved in ion selectivity and pore blocker sensitivity have been identified (Peters et al. 2015). Channel activity is required for mucus secretion induced by interleukin-13 (Lin et al. 2015; Zhang et al. 2015). Ano1 may interact cooperatively with TrpV1 (TC# 1.A.4.2.1) to form a thermal sensor (Kanazawa and Matsumoto 2014). Inhibitors have been described (Boedtkjer et al. 2015). The first intracellular loop serves as a Ca2+ binding site and includes D439, E444 and E447 (Pang et al. 2015). It is inhibited by various 4-Aryl-2-amino thiazoles at concentrations as low as 1 mμM (Piechowicz et al. 2016). ANO1 and TRPC6 (1.A.4.1.5) are present in the same macromolecular complex and localize in close spatial proximity in the myocyte plasma membrane. TRPC6 channels probably generate a local intracellular Ca2+ signal that activates nearby ANO1 channels in myocytes to stimulate vasoconstriction (Wang et al. 2016). ANO1 transports bicarbonate which functions in the regulation of pancreatic acinar cell pH (Han et al. 2016). TMEM16A contains two ion conduction pores that are independently activated by Ca2+ binding to sites that are embedded within the transmembrane part of each subunit (Lim et al. 2016). Interactions between the carboxy- terminus and the first intracellular loop in the TMEM16A homo-dimer regulate channel activity (Scudieri et al. 2016). A STAT6-TMEM16A-ERK1/2 signal pathway and TMEM16A channel activity are required for the Interleukin-13 (IL-13)-induced TMEM16A-mediated mucus production (Qin et al. 2016). Angiotensin II elicits a TMEM16A-mediated current, and TMEM16A participates in Ang II-induced basilar constriction via the RhoA/ROCK signaling pathway (Li et al. 2016). 2-acylamino-cycloalkylthiophene-3-carboxylic acid arylamides (AACTs) are inhibitors of TMEM16A, and 48 synthesized analogs (10ab-10bw) of the original AACT compound (10aa) have been synthesized and studied. The most potent compound (10bm), which contains an unusual bromodifluoroacetamide at the thiophene 2-position, had an IC50 ~ 30 nM (Truong et al. 2017). Ano1 plays a role in asthma (Wang et al. 2017). The E143A mutant showed reduced sensitivity to Ca2+ but not to high temperatures, whereas the E705V mutant exhibited reduced sensitivity to both Ca2+ and noxious heat (Choi et al. 2018). Voltage modulation of TMEM16A involves voltage-dependent occupancy of calcium- and anion-binding site(s) within the membrane electric field as well as a voltage-dependent conformational change intrinsic to the channel protein. These gating modalities all critically depend on the sixth transmembrane segment (Peters et al. 2018). TMEM16A in myocytes plays a major functional role in contraction (Mohanakumar et al. 2018). Bile acids activate TMEM16A and thereby increase cholangiocyte fluid secretion (Li et al. 2018). TMEM16A participates in H2O2-induced apoptosis via modulation of mitochondrial membrane permeability (Zeng et al. 2018). Glioma-associate oncogene proteins, Gli1 and Gli2, bind to the promoter and repress ANO1 transcription, dependent on Gli binding to a site close to the ANO1 transcriptional start site (Mazzone et al. 2019). Clarithromycin suppresses IL-13-induced goblet cell metaplasia via the TMEM16A-dependent pathway in guinea pig airway epithelial cells (Hara et al. 2019). TMEM16A is involved in gastric emptying, and TMEM16A inhibition may be effective in treating disorders of accelerated gastric emptying, such as dumping syndrome (Cil et al. 2019). Phosphatidylinositol (4,5)-bisphosphate (PIP2) regulates TMEM16A channel activation and desensitization by binding to a binding site, possibly at the cytosolic interface of TMSs 3-5. The ion-conducting pore of TMEM16A consists of two functionally distinct modules: a Ca2+-binding module formed by TMSs 6-8 and a PIP2-binding regulatory module formed by TMs 3-5, which mediate channel activation and desensitization, respectively (Sui et al. 2020). TMEM16A plays a dual role in LPS-induced intestinal epithelial barrier dysfunction (Sui et al. 2020). Hepatocyte TMEM16A plays a role in nonalcoholic fatty liver disease (NAFLD), and its deletion retards NAFLD progression by ameliorating hepatic glucose metabolic disorder (Guo et al. 2020). Hepatocyte TMEM16A interacts with vesicle-associated membrane protein 3 (VAMP3) to induce its degradation, suppressing the formation of the VAMP3/syntaxin 4 and VAMP3/synaptosome-associated protein 23 complexes (see TC# 1.F.1.1.5). This leads to impairment of hepatic glucose transporter 2 (GLUT2) translocation and glucose uptake (Guo et al. 2020). TMEM16A is a potential biomarker for Lung Cancer (Hu et al. 2019). Allosteric modulation of alternatively spliced Ca2+-activated Cl- channels, TMEM16A by PI(4,5)P2 and CaMKII (TC# 8.A.104.1.11) has been demonstrated (Ko et al. 2020). Signaling through the interleukin-4 and interleukin-13 receptor complexes regulates cholangiocyte TMEM16A expression and biliary secretion (Dutta et al. 2020). A second Ca2+ binding site allosterically controls TMEM16A activation (Le and Yang 2020). A long noncoding RNA (lncRNA), ANO1-AS2, downregulates the ANO1 gene by interacting with the ANO1 gene promoter, which can influence sperm motility and morphology (Saberiyan et al. 2020). Ano1 plays an important role in generating urethral tone (Drumm et al. 2021). Human TMEM16A shows increated expression in many cancers (Chen et al. 2021). TMEM16A is inhibitied by liquiritigenin (Kato et al. 2021) and is activated by the natural product canthaxanthin which promotes gastrointestinal contraction (Ji et al. 2020). TMEM16A ameliorates vascular remodeling by suppressing autophagy via inhibiting Bcl-2-p62 complex formation. It regulates the four-way interaction between p62 (P37198; TC# 1.I.1.1.3), Bcl-2 (TC# 1.A.21.1.10), Beclin-1 (BECN1 or GT197; Q144570; TC# 9.A.15.2.1), and VPS34 (phosphatidylinositol 3-kinase, PI 3-kinase, PIK3C3), and coordinately prevents vascular autophagy and remodeling (Lv et al. 2020). A small molecule inhibitor of TMEM16A (2-bromodifluoroacetylamino-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carbox ylic acid o-tolylamide) blocks vascular smooth muscle contraction and lowers blood pressure in spontaneously hypertensive rats (Cil et al. 2021). Evodiamine and rutecarpine are TMEM16A inhibitors (Zhao et al. 2021). Cepharanthine is a selective ANO1 inhibitor with potential for lung adenocarcinoma therapy (Zhang et al. 2021). Benzophenanthridine alkaloids suppress lung adenocarcinoma by blocking TMEM16A Ca2+-activated Cl- channels (Zhang et al. 2020). The diverse roles of TMEM16A Ca2+-activated Cl- channels in inflammation have been described (Bai et al. 2021). TMEM16A-mediated breast cancer metastasis has been described in which ROCK1 increases TMEM16A channel activity via moesin phosphorylation. An increase in TMEM16A channel activity promotes cell migration and invasion (Luo et al. 2021). Four Ca2+ sensing sites in TMEM16A have been identfied, and the activation properties of TMEM16A by them has been discussed (Ji et al. 2021). Blockade of TMEM16A protects against renal fibrosis by reducing the intracellular Cl- concentration (Li et al. 2021). The TMEM16A/anoctamin 1 inhibitor T16Ainh-A01 reverses monocrotaline-induced rat pulmonary arterial hypertension (Xie et al. 2020). The role of TMEM16A/ERK/NK-1 signaling in dorsal root ganglia neurons in the development of neuropathic pain induced by spared nerve injury has been studied (Chen et al. 2021).
Anoctamin 1a of Homo sapiens (Q5XXA6)
Anoctamin, Anoh-2. Present in mechanoreceptive neurons and spermatheca (Wang et al. 2013).
Anoh-2 of Caenorhabditis elegans
Ca-ClC Family homologue
Ca-ClC homologue of Paramecium tetraurelia (A0CAP8)
Ciliate CaClC homologue
CaClC homologue of Paramecium tetraurelia (A0CIB0)
Water mold Anoctamin-like protein
Anoctamin-like protein of Phytophthora infestans (D0NGF4)
Uncharacterized protein of Schizosaccharomyces japonicus
Anoctamin-like protein of Oxytricha trifallax
TMEM16 (Ist2) ion channel/phospholipid scramblase of 735 aas and 8 - 10 TMSs (Malvezzi et al. 2013). Three high-resolution cryo-EM structures of this scramblase, reconstituted in lipid nanodiscs, revealed that Ca2+-dependent activation of the scramblase entails global rearrangement of the transmembrane and cytosolic domains. Activation of the protein thins the membrane near the transport pathway to facilitate rapid transbilayer lipid movement (Falzone et al. 2019).
Ist2 of Aspergillus fumigatus (Neosartorya fumigata)
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).
TMEM16 of Nectria haematococca (Fusarium solani subsp. pisi)
Increased sodium tolerance protein, Ist2, of 946 aas and 7 TMSs. Ist2 is an endoplasmic reticulum (ER)-resident transmembrane protein that mediates associations between the plasma membrane (PM) and the cortical ER (cER) in baker's yeast (Kralt et al. 2015).
Ist2 of Saccharomyces cerevisiae
Anoctamin 1, isoform b (Gnathodiaphyseal dysplasia 1 protein homologue) (39% identical to Anoctamin 1a) (Planells-Cases and Jentsch, 2009). See also Xu et al. 2015.
Anoctamin 1b of Homo sapiens (Q75UR0)
Anoctamin 3, ANO3 or TMEM16C or KCNT1/Slack, of 981 aas and 9 putative TMSs. Has calcium-dependent phospholipid scramblase activity, scrambling phosphatidylcholine and galactosylceramide. Seems to act as a potassium channel regulator and may inhibit pain signaling; can facilitate KCNT1/Slack channel activity by promoting its full single-channel conductance at very low sodium concentrations and by increasing its sodium sensitivity (Scudieri et al. 2012). Mutations cause (i) epilepsy of infancy with migrating focal seizures (EIMFS; also known as migrating partial seizures in infancy), (ii) autosomal dominant nocturnal frontal lobe epilepsy, and (iii) other types of early onset epileptic encephalopathies (EOEEs) (Ohba et al. 2015). TMEM16C/Slack regulation of excitatory synaptic plasticity via GluA1-containing AMPA Receptors is critical for the pathogenesis of remifentanil-induced postoperative hyperalgesia in rats (Li et al. 2021).
ANO3 or KCNT1 of Homo sapiens
Ano5 (GDD1, TMEM16E. ) of 913 aas and 10 TMSs. Associated with bone fragility, limb girdle muscular dystrophy type 2L (LGMD2L), Miyoshi myopathy type 3 (MMD3), and gnathodiaphyseal dysplasia 1 (GDD1) in humans (Jin et al. 2017), but an Ano5 knock-out mutant in mice was not reported to exhibit such symptoms (Xu et al. 2015). The orthologue in mice is TC# 1.A.17.1.2. TMEM16E may function as a phospholipid scramblase in intracellular membranes promoting sperm motility and function (Gyobu et al. 2016). Dysregulated calcium homeostasis prevents plasma membrane repair in Anoctamin 5/TMEM16E-deficient patient muscle cells (Chandra et al. 2019). Ano5 is involved in familial florid osseous dysplasia (Lv et al. 2019). Pharmacological inhibition of ANO5 or lack of ANO5, prevent Ca2+ uptake into the ER following plasma membrane damage and Ca2+ overload (Chandra et al. 2021). Thus, Cl- uptake into the ER is required to sequester injury-promoted cytosolic Ca2+.
Ano5 of Homo sapiens
Subdued, a calcium-activated chloride channel of 1075 aas. Functions in conjunction with the thermo-TRPs in thermal nociception. Subdued channels may amplify the nociceptive neuronal firing that is initiated by thermo-TRP channels in response to thermal stimuli (Jang et al. 2015). It may also act on phospholipids to transport or hydrolyze them (Le et al. 2019).
Subdued of Drosophila melanogaster
ANO-like protein of 921 aas and 9 predicted TMSs.
ANO-L family protein of Strongylocentrotus purpuratus (Purple sea urchin)
Duplicated full length anoctamin of 2084 aas and an etimated 20 TMSs. The protein has two full length repeats, each of about 1000 aas with a ~500 aas hydrophilic domain followed by the first anoctamin domain, and then another 500 aa hydrophilic domain followed by the second anoctamin domain.
Dupicated anoctamin of Aphanomyces invadans
TMem16A or Anoctamin-1 (Ano1) Ca2+-activated anion (Cl-) channel of 960 aas and 10 TMSs. Its structure has been solved by cryoEM (Paulino et al. 2017). The protein shows a similar organization to the fungal nhTMEM16, except for changes at the site of catalysis. There, the conformation of transmembrane helices, constituting a membrane-spanning furrow that provides a path for lipids in scramblases, is replaced to form an enclosed aqueous pore that is largely shielded from the membrane (Paulino et al. 2017). It thus provides a pathway for anions such as Cl-. During activation, the binding of Ca2+ to a site located within the transmembrane domain, in the vicinity of the pore, alters the electrostatic properties of the ion conduction path and triggers a conformational rearrangement of an α-helix that comes into physical contact with the bound ligand, and thereby directly couples ligand binding and pore opening (Paulino et al. 2017). The E143A mutant showed reduced sensitivity to Ca2+ but not to high temperatures, whereas the E705V mutant exhibited reduced sensitivity to both Ca2+ and noxious heat (Choi et al. 2018). Loss of TMEM16A resulted in reduced nephron number and, subsequently, albuminuria and tubular damage (Schenk et al. 2018). mAno1 expression is regulated via alternative promoters, and its transcriptional variation results in variation of the N-terminal sequence of the Ano1 protein due to alternative translation initiation sites (Kamikawa et al. 2018).
Ano1 of Mus musculus
Anoctamin-1 or TMEM16K of 660 aas and 9 or 10 TMSs. In the presence of Ca2+, TMEM16K directly binds Ca2+ to form a stable complex (Ishihara et al. 2016). In the absence of Ca2+, TMEM16K and TMEM16F (TC# 1.A.17.1.4) aggregated, suggesting that their structures are stabilized by Ca2+. Mutagenesis of acidic residues in TMEM16K's cytoplasmic and transmembrane regions identified five residues that are critical for binding Ca2+. These residues are well conserved between TMEM16F and 16K, and point mutations of these residues in TMEM16F reduced its ability to support Ca2+-dependent phospholipid scrambling (Ishihara et al. 2016). Phosphatidyl serine in the ER of mammalian cells is predominantly localized to the cytoplasmic leaflet, but TMEM16K directly or indirectly mediates Ca2+-dependent phospholipid scrambling (Tsuji et al. 2019).
TMEM16K of Homo sapiens
Anoctamin 7, ANO7, or TMEM16G, of 933 aas and 10 TMSs. It has calcium-dependent phospholipid scramblase activity, scrambling phosphatidylserine, phosphatidylcholine and galactosylceramide, but it does not exhibit calcium-activated chloride channel (CaCC) activity. It may play a role in cell-cell interactions (Das et al. 2008). ANO7 is associated with aggressive prostate cancer (Kaikkonen et al. 2018). Insights into the topology and function of Ano7 have been described (Guo et al. 2021). Activation of calcium-activated chloride channels suppresses inherited seizure susceptibility in genetically epilepsy-prone rats (Thomas et al. 2022).
ANO7 of Homo sapiens
Anoctamin1, Ano1, TMEM16A of 979 aas and 10 TMSs. It is probably an anion (Cl-) cannel. Fertilization activates TMEM16A channels in X. laevis eggs and induces the earliest known event triggered by fertilization: the fast block to polyspermy (Wozniak et al. 2018).
Ano1 of Xenopus laivis
Anoctamin-4, ANO4, TMEM16D, of 955 aas and 10 putative TMSs. 68% identical to ANO4 (TC# 1.A.17.1.20). It has calcium-dependent phospholipid scramblase activity, scrambling phosphatidylserine, phosphatidylcholine and galactosylceramide, and it is a Ca2+-dependent non-selective cation channel (Reichhart et al. 2019). ANO4 is primarily expressed in the CNS and certain endocrine glands, and mutations affecting protein stability have been associated with various neuronal disorders (Reichhart et al. 2021).
ANO4 of Homo sapiens
TMEM16B (Anoctamin-2, ANO2) anion channel. Exists in the membrane as a homodimer where the cytoplasmic N-terminus functions in dimerization (Tien et al. 2013). TMSs 5-6 may comprise parts of the pore-loop that controls Cl- conductance (Adomaviciene et al. 2013). TMEM16A and TMEM16B are differentially expressed during development in the olfactory epithelium of the mouse (Maurya and Menini 2014).
TMEM16B of Homo sapiens (Q9NQ90)
Anoctamin-8, Ano8 or TMEM16H, of 1232 aas and 9 TMSs. It tethers the endoplasmic reticulum and plasma membrane for assembly of Ca2+ signaling complexes at the ER/PM compartment (Jha et al. 2019). ANO8 is a key tether in the formation of the ER/PM junctions that are essential for STIM1-STIM1 interaction and STIM1-Orai1 interaction and channel activation at a ER/PM PI(4,5)P2-rich compartment. Moreover, ANO8 assembles all core Ca2+ signaling proteins: Orai1, PMCA, STIM1, IP3 receptors, and SERCA2 at the ER/PM junctions to mediate a novel form of Orai1 channel inactivation by markedly facilitating SERCA2-mediated Ca2+ influx into the ER. This controls the efficiency of receptor-stimulated Ca2+ signaling, Ca2+ oscillations, and the duration of Orai1 activity to prevent Ca2+ toxicity (Jha et al. 2019).
Ano8 of Homo sapiens
Anoctamin-6 (ANO6: TMEM16F) Ca2+-dependent phospholipid scramblase (flippase) (Suzuki et al., 2010; Chauhan et al. 2016). Defects cause Scott syndrome, and promote assembly of the tenase and prothrombinase complexes involved in blood coagulation (Fujii et al. 2015). It is an essential component of the outwardly rectifying chloride channel (Martins et al., 2011; Keramidas and Lynch 2012). It has also been reported to be an anion channel with delayed Ca2+ activation (Adomaviciene et al. 2013) as well as a Ca2+-activated cation channel with activity that is required for lipid scrambing (Yang et al. 2012). However, Suzuki et al. (2013) showed that TMEM16F is a Ca2+-dependent phospholipid scramblase that exposes phosphatidylserine (PS) to the cell surface but lacks calcium-dependent chloride channel activity (see also Segawa et al. 2011). TMEM16C, 16D, 16G and 16J also have Ca2+-dependent scramblase activities but not channel activity (Suzuki et al. 2013). The pore region suggested to be resonsible for Cl- transport in TMEM16A is also responsible for phospholipid scramblase activity (Suzuki et al. 2014). Anoctamin-6 (Ano6) plays an essential role in C2C12 myoblast proliferation, probably by regulating the ERK/AKT signaling pathway (Zhao et al. 2014). It regulates baeline phosphatidyl serine exposure and cell viability in human embryonic kidney cells (Schenk et al. 2016). A single TMEM16F molecule transports phospholipids nonspecifically between the membrane bilayers dependent on Ca2+. Thermodynamic analyses indicated that TMEM16F transports 4.5 x 104 lipids per second at 25 degrees C, with an activation free energy of 47 kJ/mol, suggesting a channel-dependent, facilitated diffusion,"stepping-stone" mechanism (Watanabe et al. 2018). TMEM16F plays roles in platelet activation during blood clotting, bone formation, and T cell activation. Activation of TMEM16F by Ca2+ ionophores triggers large-scale surface membrane expansion in parallel with phospholipid scrambling (Bricogne et al. 2019). With continued ionophore application,TMEM16F-expressing cells undergo extensive shedding of ectosomes which incorporate The T cell co-receptor PD-1. Cells lacking TMEM16F fail to expand the surface membrane in response to elevated cytoplasmic Ca2+and instead undergo endocytosis with PD-1 internalization. This suggests a new role for TMEM16F as a regulator of Ca2+-activated membrane trafficking (Bricogne et al. 2a019). The inner activation gate consists of three hydrophobic residues, F518, Y563 and I612, in the middle of the phospholipid permeation pathway. Disrupting the inner gate profoundly alters phospholipid permeation. Lysine substitutions of F518 and Y563 lead to constitutively active CaPLSases that bypass Ca2+-dependent activation. An analogous lysine mutation to TMEM16F-F518 in TMEM16A (L543K) is sufficient to confer CaPLSase activity to the Ca2+-activated Cl- channel (CaCC) (Le et al. 2019). ANO6, by virtue of its scramblase activity, may play a role as a regulator of the ADAM-network in the plasma membrane. TMEM16F inhibition limits pain-associated behavior and improves motor function by promoting microglia M2 polarization in mice (Zhao and Gao 2019). Polyphenols do not inhibit the phospholipid scramblase activity of TMEM16F (Le et al. 2020).
Anoctamin-6 of Homo sapiens (Q4KMQ2)
Anoctamin-9 (Transmembrane protein 16J) (Tumor protein p53-inducible protein 5) (p53-induced gene 5 protein). It promotes pancreatic tumorigenesis (Jun et al. 2017).
ANO9 of Homo sapiens
Uncharacterized protein of Batrachochytrium dendrobatidis
Amoebozoa (Slime molds)
amoctamin-like protein of Dictyostelium purpureum
unchacterized protein of Aureococcus anophagefferens
Anoctamin, Anoh-1 of 822 aas. Functions in a sensory mode-specific manner. Present inamphid sensory neurons to detect external chemical and nociceptive cues (Wang et al. 2013).
Anoh-1 of Caenorhabditis elegans
DUF590 family protein
DUF590 protein of Dicyostelium discoideum (Q54BH1)
TMEM16 homologue of 701 aas.
TMEM16 homologue of Naegleria gruberi (Amoeba)
Anoctamin homologue of 689 aas
Anoctamin of Guillardia theta
DUF590 homologue of 487 aas
DUF590 homologue of Entamoeba nuttalli
DUF590 protein of 914 aas
DUF590 protein of Allomyces macrogynus
Uncharacterized protein of 569 aas and 8 predicted TMSs.
UP of Dictyostelium fasciculatum (Slime mold)
Uncharacterized protein of 2464 aas and 11 TMSs. Contains a trypsin-like serine protease domain (residues 100 - 400), a rabaptin (chromosome segregation) domain (residues 900 - 1200), an anoctamin domain (residues 1500 - 2000) and an AAA ATPase-containing von Willebrand factor type A domain (residues 2200 - 2500).
UP of Thalassiosira pseudonana (Marine diatom) (Cyclotella nana)
Uncharacterized protein of 1080 aas
UP of Ostreococcus lucimarinus
Anoctamin homologue of 1265 aas
Anoctamin homologue of Tetrahymena thermophila
Uncharacterized protein of 995 aas and 8 TMSs.
UP of Tetrahymena thermophila
Uncharacterized protein of 10 TMSs in a 3 + 4 +3 arrangement
UP of Paramecium tetraurelia
Uncharacterized protein of 888 aas and 10 TMSs in a 3 + 4 + 3 arrangement
UP of Paramecium tetraurelia
Uncharacterized protein of 958 aas and 11 or 12 TMSs in a 3 or 4 + 5 +3 arrangement.
UP of Paramecium tetraurelia
Uncharacterized protein of 842 aas and 9 TMSs.
UP of Thalassiosira pseudonana (Marine diatom) (Cyclotella nana)
Uncharacterized protein of 835 aas and 9 TMSs.
UP of Phytophthora parasitica (Potato buckeye rot agent)
Uncharacterized protein of 1231 aas and 9 TMSs
UP of Aureococcus anophagefferens (Harmful bloom alga)
Uncharacterized protein of 945 aas and 8 TMSs
UP of Ectocarpus siliculosus (Brown alga)
Uncharacterized protein of 1437 aas
UP of Emiliania huxleyi
Uncharacterized protein of 1150 aas
UP of Capsaspora owczarzaki
DUF590/putative methyltransferase of 1221 aas and 10 TMSs.
DUF490 homologue of Oxytricha trifallax
DUF590 homologue of 1026 aas and 10 TMSs
DUF590 homologue of Paramecium tetraurelia (ciliate)
TMC2, like TMC1, plays a role in hearing and gravity detection (Kawashima et al., 2011). Required for normal function of cochlear hair cells, possibly as a Ca2+ channel (Kim and Fettiplace 2013). TMC1 and TMC2 are both components of hair cell transduction channels and contribute to permeation properties (Pan et al. 2013; Kawashima et al. 2014). Both TMC1 and 2 interact with Protocadherin 15 (Maeda et al. 2014). TMC1 and TMC2 are components of the stereocilia mechanoelectrical transduction channel complex (Kurima et al. 2015). While TMC2 is required for mechanotransduction in mature vestibular hair cells, its expression in the immature cochlea may be an evolutionary remnant (Corns et al. 2017). Transgenic Tmc2 expression preserves inner ear hair cells and vestibular function in mice lacking Tmc1 (Asai et al. 2018). Gentamicin and other antibiotics enters neonatal mouse hair cells predominantly through sensorymMechanoelectrical transduction channels, Tmc1 and Tmc2 (Makabe et al. 2020).
TMC2 of Mus musculus (Q8R4P4)
Transmembrane channel 6, TMC6/EVER1 of 805 aas. Mutations give rise to epidermodysplasia verruciformis (EV), a rare genodermatosis, characterized by increased sensitivity to infection by the beta-subtype of human papillomaviruses (beta-HPVs), causing persistent, tinea versicolor-like dermal lesions (Horton and Stokes 2014). Biallelic mutations in either TMC6 or TMC8 are detected in more than half of the cases of the pre-malignant skin disease epidermodysplasia verruciformis (EV) which together form a complex with CIB1 (TC# 8.A.82.1.9) (Wu et al. 2020).
TMC6 of Homo sapiens
Transmembrane channel 8, TMC8/EVER2/EVIN2 of 726 aas. Mutations give rise to epidermodysplasia verruciformis (EV), a rare genodermatosis characterized by increased sensitivity to infection by the beta-subtype of human papillomaviruses (beta-HPVs) as well as increased incidence of cancer, causing persistent, tinea versicolor-like dermal lesions (Horton and Stokes 2014). This is due to release of Zn2+ and Ca2+ from the endoplasmic reticulum (Sirianant et al. 2014). The channel-like domain has been identified (Miyauchi et al. 2016). It plays a role in several aspects of human pathophysiology, such as ion channel permeability, human papillomavirus infection and skin cancer (Lu et al. 2017). Biallelic mutations in either TMC6 or TMC8 are detected in more than half of the cases of the pre-malignant skin disease epidermodysplasia verruciformis (EV), which together form a complex with CIB1 (TC# 8.A.82.1.9) in lymphocytes (Wu et al. 2020). TMC8 is a prognostic immune-associated gene in head and neck squamous cancer (HNSC) cells (Lin et al. 2021).
TMC8 of Homo sapiens
Transmembrane channel protein 3, Tmc3 of 1130 aas (Kurima et al. 2003; Beurg et al. 2014). LPS-inducible lncRNA TMC3-antisense-1 (AS1) negatively regulates the expression of IL-10 (Ye et al. 2020). In the brown planthopper, Nilaparvata lugens, TMCs is highly expressed in the female reproductive organ especially in the oviduct (Jia et al. 2020). RNAi-mediated silencing of Nltmc3 substantially decreased the egg-laying number and impaired ovary development.
Tmc3 of Mus musculus
Tmc1/Tmc2a or Tmc2b/protocadherin 15a (Pcdh15a). The complex is part of a mechanotransduction system (Maeda et al. 2014). Its trafficing to the plasma membrane depends on the transmembrane O-methyltransferase (TOMT/LRTOMT; 259 aas, 1 N-terminal TMS) (Erickson et al. 2017). Water motion is dependent on this complex (Chou et al. 2017). The role of another protein, Tmie (see TC# 8.A.115), in sensory hair cells is to target and stabilize the Tmc channel subunits to the stereocilia, the site of mechano-electrical transduction (Pacentine and Nicolson 2019). Tmc proteins 1, 2a and 2b are essential for zebrafish hearing although Tmc1 is not, probably because they can (at least partially) substitute for each other (Chen et al. 2020). There are two distinct cell types in inner ear hairs, an upper layer of teardrop shaped cells that rely on Tmc2a, and a lower layer of gourd shaped cells that rely on Tmc1/2b (Smith et al. 2020). Tmc reliance in the ear is dependent on the organ, subtype of hair cell, position within the ear, and axis of best sensitivity (Zhu et al. 2020).
Tmc1/Tmc2/Pcdh15 complex of Danio rerio (Zebrafish) (Brachydanio rerio)
Tmc4 (MBOAT7) of 712 aas and 10 TMSs (Mancina et al. 2016). Probably transports Ca2+, and other cations. May play a role in nonalcoholic fatty liver disease (NAFLD) (Sookoian et al. 2018), but it is not associated with a risk of hepatocellular carcinoma or persistent hepatitis B infection (Wang et al. 2021). Ibuprofen only minimally inhibited the taste response of the ENaC to NaCl, but it significantly inhibited the TMC4 response to NaCl with an IC50 at 1.45 mM. Thus, ibuprofen interferes with detection of salty taste via inhibition of TMC4 (Kasahara et al. 2021). This agrees with the fact that TMC4 is a chloride channel involved in high-concentration salt taste sensation (Kasahara et al. 2021). TMC4 is involved in pH and temperature-dependent modulation of salty taste (Kasahara et al. 2021). Salt-enhancing peptides were identified based on the allosteric sites in TMC4 (Shen et al. 2022).
TMC4 of Homo sapiens
The mechanoelectric-transduction (MT or MET) complex in auditory hair cells converts the mechanical stimulation of sound waves into neural signals. Tmc1 is of 760 aas and 10 TMSs and is 96% identical to mouse TMC1 (TC# 1.A.17.4.6). Novel TMC1 structural and splice variants are associated with congenital nonsyndromic deafness (Meyer et al. 2005). Variants responsible for hereditary hearing loss have been identified (Wang et al. 2018). There are varying numbers of channels per MET complex, each requiring multiple TMC1 molecules, and together operating in a coordinated, cooperative manner (Beurg et al. 2018). Ballesteros et al. 2018 generated a model of TMC1 based on X-ray and cryo-EM structures of TMEM16 proteins, revealing the presence of a large cavity near the protein-lipid interface that harbors the Beethoven mutation, suggesting that it functions as a permeation pathway. Hair cells are permeable to 3 kDa dextrans, and dextran permeation requires TMC1/2 proteins and functional MET channels (Ballesteros et al. 2018). TMC1 is a pore-forming component of MET channels in auditory and vestibular hair cells (Pan et al. 2018). KCNQ1 rescues TMC1 plasma membrane expression but not mechanosensitive channel activity (Harkcom et al. 2019). A Tmc1 mutation reduces calcium permeability and expression of MET channels in cochlear hair cells (Beurg et al. 2019). Deafness mutation D572N of TMC1 destabilizes TMC1 expression by disrupting LHFPL5 binding (Yu et al. 2020). Homozygous variants in the TMC1 and CDH23 (3354 aas and at least two TMSs, one N-terminal and one near the C-terminus; Q9H251) genes cause autosomal recessive nonsyndromic hearing loss (Zardadi et al. 2020). TMC1 forms a complex with protocadherin 15 (PCDH15, TC# 1.A.82.1.1), lipoma HMGIC fusion partner-like 5 (LHFPL5, TC# 1.A.82.1.1), and transmembrane inner ear protein (TMIE, TC# 8.A.116.1.2). Splicing isoforms of TMC1, LHFPL5, and TMIE have been identified (Zhou et al. 2021). There are four alternative splicing events for the genes encoding these three proteins. The alternative splicing of TMC1 and LHFPL5 is cochlear-specific and occurs in both neonatal and adult (mouse) cochleae (Zhou et al. 2021). Tmc1 deafness mutations impact mechanotransduction in auditory hair cells (Beurg et al. 2021). A TMC1 synonymous substitution is a variant disrupting splicing regulatory elements associated with recessive hearing loss (Vaché et al. 2021). The roles of solute carriers in auditory function have been reviewed (Qian et al. 2022). Autosomal recessive and dominant non-syndromic hearing loss can be due to pathogenic TMC1 variants (Kraatari-Tiri et al. 2022).
TMC1 of Homo sapiens
TMC5, of 1006 aas and 11 putative TMSs, promotes prostate cancer cell proliferation through cell cycle regulation and could be a target for treatment (Zhang et al. 2019).
TMC5 of Homo sapiens
Transmembrane channel-like protein 1 of 878 aas and ~ 11 TMSs (Erives and Fritzsch 2019). Mechanosensory transduction (MT) in specialized hair cells of the inner ear may be mediated by TMC1 as the pore component. Other components of the MT complex include protocadherin 15, cadherin 23, lipoma HMGIC fusion partner-like 5, transmembrane inner ear, calcium and integrin-binding family member 2, and ankyrins (Zheng and Holt 2020).
TMC1 of Amphimedon queenslandica
Transmembrane channel-like (TMC7) protein, of 723 aas and 11 TMSs. It probably transports Ca2+, and other cations. It is important for oral tongue squamous cell carcinoma (OTSCC), with rapid local invasion and metastasis. The long noncoding (lnc) RNA MIR4713HG is markedly upregulated in OTSCC. Upregulation of MIR4713HG promotes cell proliferation and metastasis (Jia et al. 2021). Micro RNA let7c5p physically binds MIR4713HG, and knockdown of let7c5p counteracts the effect of MIR4713HG on OTSCC. let7c5p exerted this role by affecting the expression level of TMC7 (Jia et al. 2021). TMC7 also affects other types (rectal and panrecatic) of cancer (Watanabe et al. 2014; Cheng et al. 2019), and may be associated with psychosis proneness (Ortega-Alonso et al. 2017).
TMC7 of Homo sapiens
Transmembrane channel-like protein-B, Tmc8 (EVER2). It occurs in the endoplasmic reticulum where it functions to release Ca2+ and Zn2+ and supresses Cl- currents (Sirianant et al. 2014). The functional variant, rs7208422 of the TMC8 gene, has been suggested to have a high impact on susceptibility to beta-papillomaviruses and their oncogenic potential and to also have an influence on alpha-type HPV-related disease (Stoehr et al. 2021).
Tmc8 of Mus musculus (Q7TN58)
Hypothetical protein, HP
HP of Salpingoeca sp. (F2U2C0)
Hypothetical protein, HP
HP of Capsaspora owczarzaki (E9C7I1)
Transmembrane channel-like protein 7, TMC7
TMC7 of Acromyrmex echinatior (F4X8H9)
Transmembrane channel-like protein-1, Tmc1. Also called Transmembrane cochlear-expressed protein-1, Beethoven protein and deafness protein. Required for normal function of cochlear hair cells, possibly as a Na+/K+/Ca2+ channel (Kim and Fettiplace 2013). TMC1 and TMC2 are both components of hair cell transduction channels and contribute to permeation properties (Pan et al. 2013; Kawashima et al. 2014). Channel activity has been demonstrated for the C. elegans orthologue, and the mouse Tmc1. The C. elegans Tmc1 is probably a Na+-activated Na+-selective mechanosensor. The C. elegans Tmc2 may be a Na+/K+ channel. The mouse Tmc1 is functional and replaces Tmc2 when expressed in C. elegans (WR Schafer, personal communication). Ca2+ currents are blocked by the peptide toxin GsMTx-4 (Beurg et al. 2014). Tmc1 and Tmc2, expressed in cochlear and vestibular hair cells, are required for hair cell mechanoelectric transduction (Nakanishi et al. 2014); mutations disrupt mechanoelectric transduction and are a cause of autosomal dominant and recessive forms of nonsyndromic hearing loss (Gao et al. 2015). Using the mutant mouse model (Tmc1; Beethoven) for progressive hearing loss in humans (DFNA36) this mutation has been shown to affect the MET channel pore, reducing its Ca2+ permeability and its affinity for the permeant blocker, dihydrostreptomycin (Corns et al. 2016). Evidence for TMC1 being the hair cell mechanosensitive channel has been evaluated (Fettiplace 2016). The human orthologue (UniProt acc # Q8TDI8) is 96% identical. Mouse LHFPL5 ((HMGIC fusion partner-like protein 5) co-expresses with TMC1 in auditory hair cells (Li et al. 2019). A region within the N-terminus of mouse TMC1 (residues 138 - 168) precludes trafficking from an intracellular location to the plasma membrane (Soler et al. 2019). TMC1 is an essential component of a leak channel that modulates tonotopy and excitability of mouse auditory hair cells (Liu et al. 2019). VRISPER/Cas has been used to correct defects that result in hereditary hearing loss (Farooq et al. 2020). Repair of Tmc1 via genetic engineering in vivo restored inner hair cell sensory transduction and hair cell morphology and transiently rescued low-frequency hearing (Yeh et al. 2020).
Tmc1 of Mus musculus
The sodium sensor/cation conductance channel activated by high extracellular Na+, Tmc-1 (Tmc1) (Chatzigeorgiou et al. 2013). It functions in salt taste chemosensation and salt avoidance and is an ionotropic sensory receptor. Wang et al. 2016 showed that C. elegans TMC-1 mediates nociceptor responses to high pH, not sodium, allowing the nematode to avoid strongly alkaline environments in which most animals cannot survive (Spalthoff and Göpfert 2016). TMC-1 and TMC-2 are required for normal egg laying in C. elegans. Mutations in these proteins cause membrane hyperpolarization and disrupt the rhythmic calcium activities in both neurons and muscles involved in egg laying. Mechanistically, TMC proteins enhance membrane depolarization through background leak currents, and ectopic expression of both C. elegans and mammalian TMC proteins results in membrane depolarization (Yue et al. 2018). TMC-1 is necessary for sodium attraction, but not aversion in the nematode. Dao et al. 2020 showed that TMC-1 contributes to the nematode's lithium induced attraction behavior, but not potassium or magnesium attraction, thus clarifying the specificity of the response. In addition, they found that sodium conditioned aversion is dependent on TMC-1 and disrupts both sodium- and lithium-induced attraction (Dao et al. 2020). The C. elegans tmc-1 is involved in egg-laying inhibition in response to harsh touch (Kaulich et al. 2021).
TMC-1 of Caenorhabditis elegans
Tmc2 channel of 1203 aas and 9 - 11 TMSs; functions in touch neurons as a mechanosensitive touch sensor (Chatzigeorgiou et al. 2013; WR Schafer, personal communication). May function as a Na+/K+ channel. TMC-1 and TMC-2 are required for normal egg laying in C. elegans. Mutations in these proteins cause membrane hyperpolarization and disrupt the rhythmic calcium activities in both neurons and muscles involved in egg laying. Mechanistically, TMC proteins enhance membrane depolarization through background leak currents, and ectopic expression of both C. elegans and mammalian TMC proteins results in membrane depolarization (Yue et al. 2018).
Tmc2 of Caenorhabditis elegans
Tmc receptor/channel of 1932 aas and about 10 TMSs. Plays a role in Drosophila proprioception and the sensory control of larval locomotion (Guo et al. 2016). These Tmc channels may be activated by membrane curvature in dendrites that are exposed to strain, possibly explaining how different cellular systems rely on a common molecular pathway for mechanosensory responses (He et al. 2019).
Animals (fruit flies)
Tmc of Drosophila melanogaster
Uncharacterized protein, DUF221, of 703 aas
UP of Zea mays
The non-rectifying, plasma membrane, calcium-permeable, stress-gated, cation channel 1 (CSC1) of 771 aas (Hou et al. 2014). Activated by hyperosmotic shock. Permeable to Ca2+, K+ and Na+. Inactivation or closure is Ca2+-dependent. The N-terminal region contains 3 TMSs, the first of which may be a cleavable signal peptide., and the C-terminal region contains 6 TMSs corresponding to the DUF221 domain. Arabidopsis contains at least 15 CSCs ((Hou et al. 2014). Some plant homologues are transcriptionally upregulated in response to vaious abiotic and biotic stresses involving mechanical perturbation (Kiyosue et al. 1994).
CSC1 of Arabidopsis thaliana
Osmotically-gated calcium conductance channel of 782 aas. CSC1 (Hou et al. 2014). Activated under hyperosmotic conditions. There are four paralogues in S. cerevisiae.
CSC1 of Saccharomyces cerevisiae
The osmosensitive calcium-permeable cation channel, CSC1 or Tmem63c, of 806 aas and ~10 TMSs. It is activated by hyperosmolarity and Ca2+ (Hou et al. 2014). Tmem63c is a potential pro-survival factor in angiotensin II-treated human podocytes (Eisenreich et al. 2020). It is regulated by microRNA-564 and transforming growth factor beta (TGFβ) in human renal cells, and is therefore a potential target for albuminuria development (Orphal et al. 2020).
CSC1 of Homo sapiens
Uncharacterized protein of 901 aas
UP of Spironucleus salmonicida
Uncharacterized protein of 1267 aas and 12 TMSs
UP of Dictyostelium discoideum (Slime mold)
Uncharacterized protein of 1548 aas and 12 TMSs.
UP of Thalassiosira pseudonana (Marine diatom) (Cyclotella nana)
Uncharacterized protein of 1172 aas
UP of Phytomonas sp. isolate EM1
Uncharacterized protein of 1258 aas and 11 TMSs.
UP of Agaricus bisporus (White button mushroom)
Csc1 homologue of 866 aas and ~ 11 TMSs. Deletioin of this gene causes C. albicans to become senstive to cations and SDS, tolerant to antifungal agents and produce filamentation (Jiang and Yang 2018).
Csc1 homologue of Candida albicans
OSCA1.2 of 772 aas and 11 TMSs. It is a dimer containing eleven TMSs per subunit, similar to other TMEM16 proteins. Jojoa Cruz et al. 2018 located the ion permeation pathway within each subunit by demonstrating that a conserved acidic residue is a determinant of channel conductance. Molecular dynamics simulations revealed membrane interactions, suggesting a role of lipids in gating. The high resolution structure of this hyperosmolality-gated calcium-permeable channel has been determined (Liu et al. 2018). It contains 11 TMSs and forms a homodimer. The pore-lining residues were clearly identified. Its cytosolic domain contains an RNA recognition motif and two unique long helices. The linker between these two helices forms an anchor in the lipid bilayer and may be essential to osmosensing. Genome-wide analyses of OSCA gene family members in Vigna radiata have revealed their involvement in the osmotic response (Yin et al. 2021).
OSCA1.2 of Arabidopsis thaliana (Mouse-ear cress)
Uncharacterized protein of 816 aas containe a DUF221 domain
UP of Danio rerio
Dimeric OSCA1.2 of 766 aas and 11 TMSs. The 3-D structure has been determined (K. Maity et al., PNAS, in press). This protein is 69% identical to the A. thaliana ortholog (TC# 1.A.17.5.10). It is a putative early stress-responsive osmolality-sensing ion channel protein. A model has been proposed by which it may mediate hyperosmolality-sensing and consequent gating of ion transport. It has a cytosolic domain structurally related to RNA recognition proteins that includes helical arms paralell to the plane of the membrane. They may sense lateral tension in the inner leaflet, caused by changes in turgor pressure, allowing gating of the channel via coupling of the two domains.
OSCA1.2 of Oryza sativa subsp. japonica (Rice)
TMEM63A or CSC1-like protein of 807 aas and ~10 TMSs. Heterozygous variants in TMEM63A have been identified as the cause of infantile-onset transient hypomyelination. TMEM63A variants are thought to cause transient hypomyelination with favorable developmental progress, but identification of a novel TMEM63A variant showed that the TMEM63A-related clinical spectrum is broad and includes severe developmental delay with seizures (Fukumura et al. 2021).
TMEM63A of Homo sapiens
Uncharacterized transmembrane protein 63B of 832 aas with a DUF221 domain.
UP of Homo sapiens
Uncharacterized transmembrane protein 63B of 832 aas with a DUF221 domain.
UP of Acanthamoeba castellanii
Uncharacterized protein of 853 aas with a DUF221 domain.
UP of Botryotinia fuckeliana
Phosphate metabolism protein 7, Phm7
Phm7 of Saccharomyces cerevisiae
Sporulation-specific protein 75, Spo75
Spo75 of Saccharomyce cerevisiae
RSN-1-like protein of 957 aas
RSN-1-like protein of Saccharomyces kudriavzevii
Early response to dehydrate stress protein, ERD4 of 785 aas. The orthologous channel protein in Dionaea muscipula may play a role in touch-induced hair calcium-electrical signals that excite the Venus flytrap (Scherzer et al. 2022).
ERD4 of Arabidopsis thaliana
Uncharacterized protein of 878 aas and 7 putative TMSs.
UP of Oxytricha trifallax
Uncharacterized protein of 707 aas and 10 TMSs
UP of Plasmodiophora brassicae
TMC-like protein 8 of 890 aas and 8 TMSs
TMC homologue of Oxytricha trifallax
Uncharacterized protein of 834 aas and 7 TMSs
UP of Oxytricha trifallax
Uncharacterized protein of 912 aas and 10 TMSs
UP of Phytophthora parasitica (Potato buckeye rot agent)
Uncharacterized protein of 620 aas and 9 TMSs
UP of Ectocarpus siliculosus (Brown alga)
Uncharacterized protein of 865 aas and 10 TMSs
UP of Guillardia theta
TMC protein of 890 aas and 10 TMSs
TMC protein of Tetrahymena thermophila
Uncharacterized protein of 1057 aas and 10 TMSs.
UP of Tetrahymena thermophila
Uncharacterized protein of 867 aas and 10 TMSs.
UP of Saprolegnia diclina
Uncharacterized protein of 836 aas and 12 TMSs.
UP of Giardia intestinalis (Giardia lamblia)
Uncharacterized protein of 637 aas and 8 TMSs.
UP of Spironucleus salmonicida
Distant Anoctamin homologue of 718 aas and 14 TMSs in a 4 + 1+1+1+2+2+2+1 arramgement.
Anoctamin homologue of Spironucleus salmonicida
Uncharacterized Anoctamin homologue of 502 aas and 8 putative TMSs
UP of Spironucleus salmonicida
Uncharacterized anoctamin homologue of 823 aas and 8 predicted TMSs in a 3 + 2 + 3 arrangement.
UP of Giardia intestinalis (Giardia lamblia)