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).

Channelopathies, defined as diseases that are caused by mutations in genes encoding ion channels, are associated with a wide variety of symptoms. 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-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.

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).

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).

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 are:

Cl- (out) ⇌ Cl- (in)


and


Cations (e.g., Ca2+) (out) ⇌ Cations (e.g., Ca2+) (in) 



This family belongs to the .

 

References:

Adomaviciene A., Smith KJ., Garnett H. and Tammaro P. (2013). Putative pore-loops of TMEM16/anoctamin channels affect channel density in cell membranes. J Physiol. 591(Pt 14):3487-505.

Almaça, J., Y. Tian, F. Aldehni, J. Ousingsawat, P. Kongsuphol, J.R. Rock, B.D. Harfe, R. Schreiber, and K. Kunzelmann. (2009). TMEM16 proteins produce volume-regulated chloride currents that are reduced in mice lacking TMEM16A. J. Biol. Chem. 284: 28571-28578.

Andra, K.K., S. Dorsey, C. Royer, and A.K. Menon. (2018). Structural mapping of fluorescently-tagged, functional nhTMEM16 scramblase in a lipid bilayer. J. Biol. Chem. [Epub: Ahead of Print]

Asai, Y., B. Pan, C. Nist-Lund, A. Galvin, A.N. Lukashkin, V.A. Lukashkina, T. Chen, W. Zhou, H. Zhu, I.J. Russell, J.R. Holt, and G.S.G. Géléoc. (2018). Transgenic Tmc2 expression preserves inner ear hair cells and vestibular function in mice lacking Tmc1. Sci Rep 8: 12124.

Ballesteros, A., C. Fenollar-Ferrer, and K.J. Swartz. (2018). Structural relationship between the putative hair cell mechanotransduction channel TMC1 and TMEM16 proteins. Elife 7:.

Bethel, N.P. and M. Grabe. (2016). Atomistic insight into lipid translocation by a TMEM16 scramblase. Proc. Natl. Acad. Sci. USA. [Epub: Ahead of Print]

Beurg, M., K.X. Kim, and R. Fettiplace. (2014). Conductance and block of hair-cell mechanotransducer channels in transmembrane channel-like protein mutants. J Gen Physiol 144: 55-69.

Beurg, M., R. Cui, A.C. Goldring, S. Ebrahim, R. Fettiplace, and B. Kachar. (2018). Variable number of TMC1-dependent mechanotransducer channels underlie tonotopic conductance gradients in the cochlea. Nat Commun 9: 2185.

Boedtkjer DM., Kim S., Jensen AB., Matchkov VM. and Andersson KE. (2015). New selective inhibitors of calcium-activated chloride channels - T16Ainh -A01, CaCCinh -A01 and MONNA - what do they inhibit? Br J Pharmacol. 172(16):4158-72.

Brunner, J.D., N.K. Lim, S. Schenck, A. Duerst, and R. Dutzler. (2014). X-ray structure of a calcium-activated TMEM16 lipid scramblase. Nature 516: 207-212.

Bulley, S., Z.P. Neeb, S.K. Burris, J.P. Bannister, C.M. Thomas-Gatewood, W. Jangsangthong, and J.H. Jaggar. (2012). TMEM16A/ANO1 Channels Contribute to the Myogenic Response in Cerebral Arteries. Circ Res 111: 1027-1036.

Caputo, A., E. Caci, L. Ferrera, N. Pedemonte, C. Barsanti, E. Sondo, U. Pfeffer, R. Ravazzolo, O. Zegarra-Moran, and L.J. Galietta. (2008). TMEM16A, a membrane protein associated with calcium-dependent chloride channel activity. Science 322: 590-594.

Chatzigeorgiou, M., S. Bang, S.W. Hwang, and W.R. Schafer. (2013). tmc-1 encodes a sodium-sensitive channel required for salt chemosensation in C. elegans. Nature 494: 95-99.

Chauhan, N., L. Farine, K. Pandey, A.K. Menon, and P. Bütikofer. (2016). Lipid topogenesis - 35years on. Biochim. Biophys. Acta. [Epub: Ahead of Print]

Chen, Y., H. An, T. Li, Y. Liu, C. Gao, P. Guo, H. Zhang, and Y. Zhan. (2011). Direct or indirect regulation of calcium-activated chloride channel by calcium. J. Membr. Biol. 240: 121-129.

Choi, J., Y. Jang, H. Kim, J. Wee, S. Cho, W.S. Son, S.M. Kim, and Y.D. Yang. (2018). Functional roles of glutamic acid E143 and E705 residues in the N-terminus and transmembrane domain 7 of Anoctamin 1 in calcium and noxious heat sensing. BMB Rep. [Epub: Ahead of Print]

Chou, S.W., Z. Chen, S. Zhu, R.W. Davis, J. Hu, L. Liu, C.A. Fernando, K. Kindig, W.C. Brown, R. Stepanyan, and B.M. McDermott, Jr. (2017). A molecular basis for water motion detection by the mechanosensory lateral line of zebrafish. Nat Commun 8: 2234.

Corey, D.P. and J.R. Holt. (2016). Are TMCs the Mechanotransduction Channels of Vertebrate Hair Cells? J. Neurosci. 36: 10921-10926.

Corns, L.F., J.Y. Jeng, G.P. Richardson, C.J. Kros, and W. Marcotti. (2017). TMC2 Modifies Permeation Properties of the Mechanoelectrical Transducer Channel in Early Postnatal Mouse Cochlear Outer Hair Cells. Front Mol Neurosci 10: 326.

Corns, L.F., S.L. Johnson, C.J. Kros, and W. Marcotti. (2016). Tmc1 Point Mutation Affects Ca2+ Sensitivity and Block by Dihydrostreptomycin of the Mechanoelectrical Transducer Current of Mouse Outer Hair Cells. J. Neurosci. 36: 336-349.

Dang, S., S. Feng, J. Tien, C.J. Peters, D. Bulkley, M. Lolicato, J. Zhao, K. Zuberbühler, W. Ye, L. Qi, T. Chen, C.S. Craik, Y. Nung Jan, D.L. Minor, Jr, Y. Cheng, and L. Yeh Jan. (2017). Cryo-EM structures of the TMEM16A calcium-activated chloride channel. Nature. [Epub: Ahead of Print]

Das, S., Y. Hahn, D.A. Walker, S. Nagata, M.C. Willingham, D.M. Peehl, T.K. Bera, B. Lee, and I. Pastan. (2008). Topology of NGEP, a prostate-specific cell:cell junction protein widely expressed in many cancers of different grade level. Cancer Res 68: 6306-6312.

Duran, C. and H.C. Hartzell. (2011). Physiological roles and diseases of Tmem16/Anoctamin proteins: are they all chloride channels? Acta Pharmacol Sin 32: 685-692.

Erickson, T., C.P. Morgan, J. Olt, K. Hardy, E. Busch-Nentwich, R. Maeda, R. Clemens, J.F. Krey, A. Nechiporuk, P.G. Barr-Gillespie, W. Marcotti, and T. Nicolson. (2017). Integration of Tmc1/2 into the mechanotransduction complex in zebrafish hair cells is regulated by Transmembrane O-methyltransferase (Tomt). Elife 6:.

Ferrera, L., A. Caputo, I. Ubby, E. Bussani, O. Zegarra-Moran, R. Ravazzolo, F. Pagani, and L.J. Galietta. (2009). Regulation of TMEM16A chloride channel properties by alternative splicing. J. Biol. Chem. 284: 33360-33368.

Fettiplace, R. (2016). Is TMC1 the Hair Cell Mechanotransducer Channel? Biophys. J. 111: 3-9.

Fujii, T., A. Sakata, S. Nishimura, K. Eto, and S. Nagata. (2015). TMEM16F is required for phosphatidylserine exposure and microparticle release in activated mouse platelets. Proc. Natl. Acad. Sci. USA 112: 12800-12805.

Galietta, L.J. (2009). The TMEM16 protein family: a new class of chloride channels? Biophys. J. 97: 3047-3053.

Gao X., Huang SS., Yuan YY., Wang GJ., Xu JC., Ji YB., Han MY., Yu F., Kang DY., Lin X. and Dai P. (2015). Targeted gene capture and massively parallel sequencing identify TMC1 as the causative gene in a six-generation Chinese family with autosomal dominant hearing loss. Am J Med Genet A. 167A(10):2357-65.

Gui D., Li Y. and Chen X. (2015). Alterations of TMEM16a allostery in human retinal microarterioles in long-standing hypertension. IUBMB Life. 67(5):348-54.

Guo, Y., Y. Wang, W. Zhang, S. Meltzer, D. Zanini, Y. Yu, J. Li, T. Cheng, Z. Guo, Q. Wang, J.S. Jacobs, Y. Sharma, D.F. Eberl, M.C. Göpfert, L.Y. Jan, Y.N. Jan, and Z. Wang. (2016). Transmembrane channel-like (tmc) gene regulates Drosophila larval locomotion. Proc. Natl. Acad. Sci. USA. [Epub: Ahead of Print]

Gyobu, S., H. Miyata, M. Ikawa, D. Yamazaki, H. Takeshima, J. Suzuki, and S. Nagata. (2016). A Role of TMEM16E Carrying a Scrambling Domain in Sperm Motility. Mol. Cell Biol. 36: 645-659.

Gyobu, S., K. Ishihara, J. Suzuki, K. Segawa, and S. Nagata. (2017). Characterization of the scrambling domain of the TMEM16 family. Proc. Natl. Acad. Sci. USA 114: 6274-6279.

Han, Y., A.M. Shewan, and P. Thorn. (2016). HCO3- transport through anoctamin/transmembrane protein ANO1/TMEM16A, in pancreatic acinar cells, regulates luminal pH. J. Biol. Chem. [Epub: Ahead of Print]

Horton, J.S. and A.J. Stokes. (2014). The transmembrane channel-like protein family and human papillomaviruses: Insights into epidermodysplasia verruciformis and progression to squamous cell carcinoma. Oncoimmunology 3: e28288.

Hou, C., W. Tian, T. Kleist, K. He, V. Garcia, F. Bai, Y. Hao, S. Luan, and L. Li. (2014). DUF221 proteins are a family of osmosensitive calcium-permeable cation channels conserved across eukaryotes. Cell Res 24: 632-635.

Huang, F., J.R. Rock, B.D. Harfe, T. Cheng, X. Huang, Y.N. Jan, and L.Y. Jan. (2009). Studies on expression and function of the TMEM16A calcium-activated chloride channel. Proc. Natl. Acad. Sci. USA 106: 21413-21418.

Huang, F., X. Wong, and L.Y. Jan. (2012). International Union of Basic and Clinical Pharmacology. LXXXV: calcium-activated chloride channels. Pharmacol Rev 64: 1-15.

Hwang, S.J., P.J. Blair, F.C. Britton, K.E. O'Driscoll, G. Hennig, Y.R. Bayguinov, J.R. Rock, B.D. Harfe, K.M. Sanders, and S.M. Ward. (2009). Expression of anoctamin 1/TMEM16A by interstitial cells of Cajal is fundamental for slow wave activity in gastrointestinal muscles. J. Physiol. 587: 4887-4904.

Ishihara, K., J. Suzuki, and S. Nagata. (2016). Role of Ca2+ in the Stability and Function of TMEM16F and 16K. Biochemistry 55: 3180-3188.

Jang, W., J.Y. Kim, S. Cui, J. Jo, B.C. Lee, Y. Lee, K.S. Kwon, C.S. Park, and C. Kim. (2015). The anoctamin family channel subdued mediates thermal nociception in Drosophila. J. Biol. Chem. 290: 2521-2528.

Jeon, J.H., S.S. Paik, M.H. Chun, U. Oh, and I.B. Kim. (2013). Presynaptic Localization and Possible Function of Calcium-Activated Chloride Channel Anoctamin 1 in the Mammalian Retina. PLoS One 8: e67989.

Jiang, L. and Y. Yang. (2018). The putative transient receptor potential (TRP) channel protein encoded by the orf19.4805 is involved in cation sensitivity, antifungal tolerance and filamentation in Candida albicans. Can. J. Microbiol. [Epub: Ahead of Print]

Jin, L., Y. Liu, F. Sun, M.T. Collins, K. Blackwell, A.S. Woo, E.J. Reichenberger, and Y. Hu. (2017). Three novel ANO5 missense mutations in Caucasian and Chinese families and sporadic cases with gnathodiaphyseal dysplasia. Sci Rep 7: 40935.

Jojoa Cruz, S., K. Saotome, S.E. Murthy, C.C.A. Tsui, M.S. Sansom, A. Patapoutian, and A.B. Ward. (2018). Cryo-EM structure of the mechanically activated ion channel OSCA1.2. Elife 7:. [Epub: Ahead of Print]

Jun, I., H.S. Park, H. Piao, J.W. Han, M.J. An, B.G. Yun, X. Zhang, Y.H. Cha, Y.K. Shin, J.I. Yook, J. Jung, H.Y. Gee, J.S. Park, D.S. Yoon, H.C. Jeung, and M.G. Lee. (2017). ANO9/TMEM16J promotes tumourigenesis via EGFR and is a novel therapeutic target for pancreatic cancer. Br J Cancer 117: 1798-1809.

Jung J., Nam JH., Park HW., Oh U., Yoon JH. and Lee MG. (2013). Dynamic modulation of ANO1/TMEM16A HCO3(-) permeability by Ca2+/calmodulin. Proc Natl Acad Sci U S A. 110(1):360-5.

Kamikawa, A., J. Sakazaki, and O. Ichii. (2018). Tissue-specific variation in 5''-terminal exons of mouse Anoctamin 1 transcript induces N-terminal variation of its protein via alternative translational start sites. Biochem. Biophys. Res. Commun. 503: 1710-1715.

Kanazawa, T. and S. Matsumoto. (2014). Expression of transient receptor potential vanilloid 1 and anoctamin 1 in rat trigeminal ganglion neurons innervating the tongue. Brain Res Bull 106: 17-20.

Kawashima Y., Kurima K., Pan B., Griffith AJ. and Holt JR. (2015). Transmembrane channel-like (TMC) genes are required for auditory and vestibular mechanosensation. Pflugers Arch. 467(1):85-94.

Kawashima, Y., G.S. Géléoc, K. Kurima, V. Labay, A. Lelli, Y. Asai, T. Makishima, D.K. Wu, C.C. Della Santina, J.R. Holt, and A.J. Griffith. (2011). Mechanotransduction in mouse inner ear hair cells requires transmembrane channel-like genes. J Clin Invest 121: 4796-4809.

Keramidas A. and Lynch JW. (2013). An outline of desensitization in pentameric ligand-gated ion channel receptors. Cell Mol Life Sci. 70(7):1241-53.

Kim, K.X. and R. Fettiplace. (2013). Developmental changes in the cochlear hair cell mechanotransducer channel and their regulation by transmembrane channel-like proteins. J Gen Physiol 141: 141-148.

Kim, K.X., M. Beurg, C.M. Hackney, D.N. Furness, S. Mahendrasingam, and R. Fettiplace. (2013). The role of transmembrane channel-like proteins in the operation of hair cell mechanotransducer channels. J Gen Physiol 142: 493-505.

Kiyosue, T., K. Yamaguchi-Shinozaki, and K. Shinozaki. (1994). ERD15, a cDNA for a dehydration-induced gene from Arabidopsis thaliana. Plant Physiol. 106: 1707.

Kralt, A., M. Carretta, M. Mari, F. Reggiori, A. Steen, B. Poolman, and L.M. Veenhoff. (2015). Intrinsically disordered linker and plasma membrane-binding motif sort Ist2 and Ssy1 to junctions. Traffic 16: 135-147.

Kumar, S., W. Namkung, A.S. Verkman, and P.K. Sharma. (2012). Novel 5-substituted benzyloxy-2-arylbenzofuran-3-carboxylic acids as calcium activated chloride channel inhibitors. Bioorg Med Chem 20: 4237-4244.

Kunzelmann, K., I. Cabrita, P. Wanitchakool, J. Ousingsawat, L. Sirianant, R. Benedetto, and R. Schreiber. (2015). Modulating Ca2+ signals: a common theme for TMEM16, Ist2, and TMC. Pflugers Arch. [Epub: Ahead of Print]

Kurima, K., S. Ebrahim, B. Pan, M. Sedlacek, P. Sengupta, B.A. Millis, R. Cui, H. Nakanishi, T. Fujikawa, Y. Kawashima, B.Y. Choi, K. Monahan, J.R. Holt, A.J. Griffith, and B. Kachar. (2015). TMC1 and TMC2 Localize at the Site of Mechanotransduction in Mammalian Inner Ear Hair Cell Stereocilia. Cell Rep 12: 1606-1617.

Kurima, K., Y. Yang, K. Sorber, and A.J. Griffith. (2003). Characterization of the transmembrane channel-like (TMC) gene family: functional clues from hearing loss and epidermodysplasia verruciformis. Genomics 82: 300-308.

Labay, V., R.M. Weichert, T. Makishima, and A.J. Griffith. (2010). Topology of transmembrane channel-like gene 1 protein. Biochemistry 49: 8592-8598.

Li, Q., A. Dutta, C. Kresge, A. Bugde, and A.P. Feranchak. (2018). Bile acids stimulate cholangiocyte fluid secretion by activation of transmembrane member 16A Cl channels. Hepatology. [Epub: Ahead of Print]

Li, R.S., Y. Wang, H.S. Chen, F.Y. Jiang, Q. Tu, W.J. Li, and R.X. Yin. (2016). TMEM16A contributes to angiotensin II-induced cerebral vasoconstriction via the RhoA/ROCK signaling pathway. Mol Med Rep 13: 3691-3699.

Lim, N.K., A.K. Lam, and R. Dutzler. (2016). Independent activation of ion conduction pores in the double-barreled calcium-activated chloride channel TMEM16A. J Gen Physiol 148: 375-392.

Lin J., Jiang Y., Li L., Liu Y., Tang H. and Jiang D. (2015). TMEM16A mediates the hypersecretion of mucus induced by Interleukin-13. Exp Cell Res. 334(2):260-9.

Liu J., Liu Y., Ren Y., Kang L. and Zhang L. (2014). Transmembrane protein with unknown function 16A overexpression promotes glioma formation through the nuclear factor-kappaB signaling pathway. Mol Med Rep. 9(3):1068-74.

Liu, X., J. Wang, and L. Sun. (2018). Structure of the hyperosmolality-gated calcium-permeable channel OSCA1.2. Nat Commun 9: 5060.

Loewen, M.E. and G.W. Forsyth. (2005). Structure and function of CLCA proteins. Physiol. Rev. 85: 1061-1092.

Lu, P., Q. Ding, S. Ding, Y. Fan, X. Li, D. Tian, and M. Liu. (2017). Transmembrane channel-like protein 8 as a potential biomarker for poor prognosis of hepatocellular carcinoma. Mol Clin Oncol 7: 244-248.

Maeda, R., K.S. Kindt, W. Mo, C.P. Morgan, T. Erickson, H. Zhao, R. Clemens-Grisham, P.G. Barr-Gillespie, and T. Nicolson. (2014). Tip-link protein protocadherin 15 interacts with transmembrane channel-like proteins TMC1 and TMC2. Proc. Natl. Acad. Sci. USA 111: 12907-12912.

Mancina, R.M., P. Dongiovanni, S. Petta, P. Pingitore, M. Meroni, R. Rametta, J. Borén, T. Montalcini, A. Pujia, O. Wiklund, G. Hindy, R. Spagnuolo, B.M. Motta, R.M. Pipitone, A. Craxì, S. Fargion, V. Nobili, P. Käkelä, V. Kärjä, V. Männistö, J. Pihlajamäki, D.F. Reilly, J. Castro-Perez, J. Kozlitina, L. Valenti, and S. Romeo. (2016). The MBOAT7-TMC4 Variant rs641738 Increases Risk of Nonalcoholic Fatty Liver Disease in Individuals of European Descent. Gastroenterology. [Epub: Ahead of Print]

Manji, S.S., K.A. Miller, L.H. Williams, and H.H. Dahl. (2012). Identification of three novel hearing loss mouse strains with mutations in the Tmc1 gene. Am J Pathol 180: 1560-1569.

Martins, J.R., D. Faria, P. Kongsuphol, B. Reisch, R. Schreiber, and K. Kunzelmann. (2011). Anoctamin 6 is an essential component of the outwardly rectifying chloride channel. Proc. Natl. Acad. Sci. USA 108: 18168-18172.

Maurya, D.K. and A. Menini. (2014). Developmental expression of the calcium-activated chloride channels TMEM16A and TMEM16B in the mouse olfactory epithelium. Dev Neurobiol 74: 657-675.

Medrano-Soto, A., G. Moreno-Hagelsieb, D. McLaughlin, Z.S. Ye, K.J. Hendargo, and M.H. Saier, Jr. (2018). Bioinformatic characterization of the Anoctamin Superfamily of Ca2+-activated ion channels and lipid scramblases. PLoS One 13: e0192851.

Milenkovic, V.M., M. Brockmann, H. Stöhr, B.H. Weber, and O. Strauss. (2010). Evolution and functional divergence of the anoctamin family of membrane proteins. BMC Evol Biol 10: 319.

Miyauchi, T., T. Nomura, S. Suzuki, M. Takeda, S. Shinkuma, K. Arita, Y. Fujita, and H. Shimizu. (2016). Genetic analysis of a novel splice-site mutation in TMC8 reveals the in vivo importance of the transmembrane channel-like domain of TMC8. Br J Dermatol. [Epub: Ahead of Print]

Mohanakumar, S., J. Majgaard, N. Telinius, N. Katballe, E. Pahle, V.E. Hjortdal, and D.M.B. Boedtkjer. (2018). Spontaneous and α-adrenoceptor-induced contractility in human collecting lymphatic vessels require chloride. Am. J. Physiol. Heart Circ Physiol. [Epub: Ahead of Print]

Mroz, M.S. and S.J. Keely. (2012). Epidermal growth factor chronically upregulates Ca2+-dependent Cl- conductance and TMEM16A expression in intestinal epithelial cells. J. Physiol. 590: 1907-1920.

Nakanishi H., Kurima K., Kawashima Y. and Griffith AJ. (2014). Mutations of TMC1 cause deafness by disrupting mechanoelectrical transduction. Auris Nasus Larynx. 41(5):399-408.

Ni, Y.L., A.S. Kuan, and T.Y. Chen. (2014). Activation and inhibition of TMEM16A calcium-activated chloride channels. PLoS One 9: e86734.

Oh, U. and J. Jung. (2016). Cellular functions of TMEM16/anoctamin. Pflugers Arch 468: 443-453.

Ohba, C., M. Kato, N. Takahashi, H. Osaka, T. Shiihara, J. Tohyama, S. Nabatame, J. Azuma, Y. Fujii, M. Hara, R. Tsurusawa, T. Inoue, R. Ogata, Y. Watanabe, N. Togashi, H. Kodera, M. Nakashima, Y. Tsurusaki, N. Miyake, F. Tanaka, H. Saitsu, and N. Matsumoto. (2015). De novo KCNT1 mutations in early-onset epileptic encephalopathy. Epilepsia 56: e121-128.

Ousingsawat, J., J.R. Martins, R. Schreiber, J.R. Rock, B.D. Harfe, and K. Kunzelmann. (2009). Loss of TMEM16A causes a defect in epithelial Ca2+-dependent chloride transport. J. Biol. Chem. 284: 28698-28703.

Pan, B., G.S. Géléoc, Y. Asai, G.C. Horwitz, K. Kurima, K. Ishikawa, Y. Kawashima, A.J. Griffith, and J.R. Holt. (2013). TMC1 and TMC2 Are Components of the Mechanotransduction Channel in Hair Cells of the Mammalian Inner Ear. Neuron. 79: 504-515.

Pan, B., N. Akyuz, X.P. Liu, Y. Asai, C. Nist-Lund, K. Kurima, B.H. Derfler, B. György, W. Limapichat, S. Walujkar, L.N. Wimalasena, M. Sotomayor, D.P. Corey, and J.R. Holt. (2018). TMC1 Forms the Pore of Mechanosensory Transduction Channels in Vertebrate Inner Ear Hair Cells. Neuron. 99: 736-753.e6.

Pang C., Yuan H., Ren S., Chen Y., An H. and Zhan Y. (201). TMEM16A/B associated CaCC: structural and functional insights. Protein Pept Lett. 21(1):94-9.

Pang, C.L., H.B. Yuan, T.G. Cao, J.G. Su, Y.F. Chen, H. Liu, H. Yu, H.L. Zhang, Y. Zhan, H.L. An, and Y.B. Han. (2015). Molecular simulation assisted identification of Ca2+ binding residues in TMEM16A. J Comput Aided Mol Des. [Epub: Ahead of Print]

Paulino, C., V. Kalienkova, A.K.M. Lam, Y. Neldner, and R. Dutzler. (2017). Activation mechanism of the calcium-activated chloride channel TMEM16A revealed by cryo-EM. Nature. [Epub: Ahead of Print]

Paulino, C., Y. Neldner, A.K. Lam, V. Kalienkova, J.D. Brunner, S. Schenck, and R. Dutzler. (2017). Structural basis for anion conduction in the calcium-activated chloride channel TMEM16A. Elife 6:.

Peters CJ., Yu H., Tien J., Jan YN., Li M. and Jan LY. (2015). Four basic residues critical for the ion selectivity and pore blocker sensitivity of TMEM16A calcium-activated chloride channels. Proc Natl Acad Sci U S A. 112(11):3547-52.

Peters, C.J., J.M. Gilchrist, J. Tien, N.P. Bethel, L. Qi, T. Chen, L. Wang, Y.N. Jan, M. Grabe, and L.Y. Jan. (2018). The Sixth Transmembrane Segment Is a Major Gating Component of the TMEM16A Calcium-Activated Chloride Channel. Neuron. [Epub: Ahead of Print]

Piechowicz, K.A., E.C. Truong, K.M. Javed, R.R. Chaney, J.Y. Wu, P.W. Phuan, A.S. Verkman, and M.O. Anderson. (2016). Synthesis and evaluation of 5,6-disubstituted thiopyrimidine aryl aminothiazoles as inhibitors of the calcium-activated chloride channel TMEM16A/Ano1. J Enzyme Inhib Med Chem 1-7. [Epub: Ahead of Print]

Planells-Cases, R. and T.J. Jentsch. (2009). Chloride channelopathies. Biochim. Biophys. Acta. [Epub: Ahead of Print]

Qin, Y., Y. Jiang, A.S. Sheikh, S. Shen, J. Liu, and D. Jiang. (2016). Interleukin-13 stimulates MUC5AC expression via a STAT6-TMEM16A-ERK1/2 pathway in human airway epithelial cells. Int Immunopharmacol 40: 106-114.

Schenk, L.K., B. Buchholz, S.F. Henke, U. Michgehl, C. Daniel, K. Amann, K. Kunzelmann, and H.J. Pavenstädt. (2018). Nephron-specific knockout of TMEM16A leads to reduced number of glomeruli and albuminuria. Am. J. Physiol. Renal Physiol. [Epub: Ahead of Print]

Schenk, L.K., U. Schulze, S. Henke, T. Weide, and H. Pavenstädt. (2016). TMEM16F Regulates Baseline Phosphatidylserine Exposure and Cell Viability in Human Embryonic Kidney Cells. Cell Physiol Biochem 38: 2452-2463.

Schreiber, R., I. Uliyakina, P. Kongsuphol, R. Warth, M. Mirza, J.R. Martins, and K. Kunzelmann. (2010). Expression and function of epithelial anoctamins. J. Biol. Chem. 285: 7838-7845.

Scudieri, P., E. Sondo, L. Ferrera, and L.J. Galietta. (2012). The anoctamin family: TMEM16A and TMEM16B as calcium-activated chloride channels. Exp Physiol 97: 177-183.

Scudieri, P., I. Musante, A. Gianotti, O. Moran, and L.J. Galietta. (2016). Intermolecular Interactions in the TMEM16A Dimer Controlling Channel Activity. Sci Rep 6: 38788.

Segawa, K., J. Suzuki, and S. Nagata. (2011). Constitutive exposure of phosphatidylserine on viable cells. Proc. Natl. Acad. Sci. USA 108: 19246-19251.

Shimizu, T., T. Iehara, K. Sato, T. Fujii, H. Sakai, and Y. Okada. (2013). TMEM16F is a component of a Ca2+-activated Cl- channel but not a volume-sensitive outwardly rectifying Cl- channel. Am. J. Physiol. Cell Physiol. 304: C748-759.

Shiwarski, D.J., C. Shao, A. Bill, J. Kim, D. Xiao, C.A. Bertrand, R.S. Seethala, D. Sano, J.N. Myers, P. Ha, J. Grandis, L.A. Gaither, M.A. Puthenveedu, and U. Duvvuri. (2014). To "Grow" or "Go": TMEM16A Expression as a Switch between Tumor Growth and Metastasis in SCCHN. Clin Cancer Res 20: 4673-4688.

Sirianant L., Ousingsawat J., Tian Y., Schreiber R. and Kunzelmann K. (2014). TMC8 (EVER2) attenuates intracellular signaling by Zn2+ and Ca2+ and suppresses activation of Cl- currents. Cell Signal. 26(12):2826-33.

Smith, C.J., J.D. Watson, W.C. Spencer, T. O'Brien, B. Cha, A. Albeg, M. Treinin, and D.M. Miller, 3rd. (2010). Time-lapse imaging and cell-specific expression profiling reveal dynamic branching and molecular determinants of a multi-dendritic nociceptor in C. elegans. Dev Biol 345: 18-33.

Sookoian, S., D. Flichman, M.E. Garaycoechea, C. Gazzi, J.S. Martino, G.O. Castaño, and C.J. Pirola. (2018). Lack of evidence supporting a role of TMC4-rs641738 missense variant-MBOAT7- intergenic downstream variant-in the Susceptibility to Nonalcoholic Fatty Liver Disease. Sci Rep 8: 5097.

Spalthoff, C. and M.C. Göpfert. (2016). Sensing pH with TMCs. Neuron. 91: 6-8.

Suzuki T., Suzuki J. and Nagata S. (2014). Functional swapping between transmembrane proteins TMEM16A and TMEM16F. J Biol Chem. 289(11):7438-47.

Suzuki, J., M. Umeda, P.J. Sims, and S. Nagata. (2010). Calcium-dependent phospholipid scrambling by TMEM16F. Nature 468: 834-838.

Suzuki, J., T. Fujii, T. Imao, K. Ishihara, H. Kuba, and S. Nagata. (2013). Calcium-dependent Phospholipid Scramblase Activity of TMEM16 Protein Family Members. J. Biol. Chem. 288: 13305-13316.

Tien, J., H.Y. Lee, D.L. Minor, Jr, Y.N. Jan, and L.Y. Jan. (2013). Identification of a dimerization domain in the TMEM16A calcium-activated chloride channel (CaCC). Proc. Natl. Acad. Sci. USA 110: 6352-6357.

Truong, E.C., P.W. Phuan, A.L. Reggi, L. Ferrera, L.J.V. Galietta, S.E. Levy, A.C. Moises, O. Cil, E. Diez-Cecilia, S. Lee, A.S. Verkman, and M.O. Anderson. (2017). Substituted 2-acylamino-cycloalkylthiophene-3-carboxylic acid arylamides as inhibitors of the calcium-activated chloride channel transmembrane protein 16A (TMEM16A). J Med Chem. [Epub: Ahead of Print]

Wang Y., Alam T., Hill-Harfe K., Lopez AJ., Leung CK., Iribarne D., Bruggeman B., Miyamoto MM., Harfe BD. and Choe KP. (2013). Phylogenetic, expression, and functional analyses of anoctamin homologs in Caenorhabditis elegans. Am J Physiol Regul Integr Comp Physiol. 305(11):R1376-89.

Wang, H., K. Wu, J. Guan, J. Yang, L. Xie, F. Xiong, L. Lan, D. Wang, and Q. Wang. (2018). Identification of four TMC1 variations in different Chinese families with hereditary hearing loss. Mol Genet Genomic Med. [Epub: Ahead of Print]

Wang, L., Y. Iwasaki, K.K. Andra, K. Pandey, A.K. Menon, and P. Bütikofer. (2018). Scrambling of natural and fluorescently tagged phosphatidylinositol by reconstituted G protein-coupled receptor and TMEM16 scramblases. J. Biol. Chem. [Epub: Ahead of Print]

Wang, P., W. Zhao, J. Sun, T. Tao, X. Chen, Y.Y. Zheng, C.H. Zhang, Z. Chen, Y.Q. Gao, F. She, Y.Q. Li, L.S. Wei, P. Lu, C.P. Chen, J. Zhou, D.Q. Wang, L. Chen, X.H. Shi, L. Deng, R. ZhuGe, H.Q. Chen, and M.S. Zhu. (2017). Inflammatory mediators mediate airway smooth muscle contraction through a G protein-coupled receptor-transmembrane protein 16A-voltage-dependent Ca2+ channel axis and contribute to bronchial hyperresponsiveness in asthma. J Allergy Clin Immunol. [Epub: Ahead of Print]

Wang, Q., M.D. Leo, D. Narayanan, K.P. Kuruvilla, and J.H. Jaggar. (2016). Local coupling of TRPC6 to ANO1/TMEM16A channels in smooth muscle cells amplifies vasoconstriction in cerebral arteries. Am. J. Physiol. Cell Physiol. 310: C1001-1009.

Wang, X., G. Li, J. Liu, J. Liu, and X.Z. Xu. (2016). TMC-1 Mediates Alkaline Sensation in C. elegans through Nociceptive Neuron.s. Neuron. 91: 146-154.

Watanabe, R., T. Sakuragi, H. Noji, and S. Nagata. (2018). Single-molecule analysis of phospholipid scrambling by TMEM16F. Proc. Natl. Acad. Sci. USA 115: 3066-3071.

Winkler M., Kuhner P., Russ U., Ortiz D., Bryan J. and Quast U. (2012). Role of the amino-terminal transmembrane domain of sulfonylurea receptor SUR2B for coupling to K(IR)6.2, ligand binding, and oligomerization. Naunyn Schmiedebergs Arch Pharmacol. 385(3):287-98.

Winpenny, J.P., L.L. Marsey, and D.W. Sexton. (2009). The CLCA gene family: putative therapeutic target for respiratory diseases. Inflamm Allergy Drug Targets 8: 146-160.

Wozniak, K.L., W.A. Phelps, M. Tembo, M.T. Lee, and A.E. Carlson. (2018). The TMEM16A channel mediates the fast polyspermy block in. J Gen Physiol. [Epub: Ahead of Print]

Wu, Z., N. Grillet, B. Zhao, C. Cunningham, S. Harkins-Perry, B. Coste, S. Ranade, N. Zebarjadi, M. Beurg, R. Fettiplace, A. Patapoutian, and U. Müller. (2016). Mechanosensory hair cells express two molecularly distinct mechanotransduction channels. Nat Neurosci. [Epub: Ahead of Print]

Xu, J., M. El Refaey, L. Xu, L. Zhao, Y. Gao, K. Floyd, T. Karaze, P.M. Janssen, and R. Han. (2015). Genetic disruption of Ano5 in mice does not recapitulate human ANO5-deficient muscular dystrophy. Skelet Muscle 5: 43.

Yang, H., A. Kim, T. David, D. Palmer, T. Jin, J. Tien, F. Huang, T. Cheng, S.R. Coughlin, Y.N. Jan, and L.Y. Jan. (2012). TMEM16F forms a Ca2+-activated cation channel required for lipid scrambling in platelets during blood coagulation. Cell 151: 111-122.

Yang, T., W.A. Hendrickson, and H.M. Colecraft. (2014). Preassociated apocalmodulin mediates Ca2+-dependent sensitization of activation and inactivation of TMEM16A/16B Ca2+-gated Cl- channels. Proc. Natl. Acad. Sci. USA 111: 18213-18218.

Yang, Y.D., H. Cho, J.Y. Koo, M.H. Tak, Y. Cho, W.S. Shim, S.P. Park, J. Lee, B. Lee, B.M. Kim, R. Raouf, Y.K. Shin, and U. Oh. (2008). TMEM16A confers receptor-activated calcium-dependent chloride conductance. Nature 455: 1210-1215.

Yue, X., J. Zhao, X. Li, Y. Fan, D. Duan, X. Zhang, W. Zou, Y. Sheng, T. Zhang, Q. Yang, J. Luo, S. Duan, R. Xiao, and L. Kang. (2018). TMC Proteins Modulate Egg Laying and Membrane Excitability through a Background Leak Conductance in C. elegans. Neuron. 97: 571-585.e5.

Zeng, J.W., B.Y. Chen, X.F. Lv, L. Sun, X.L. Zeng, H.Q. Zheng, Y.H. Du, G.L. Wang, M.M. Ma, and Y.Y. Guan. (2018). TMEM16A Participates in Hydrogen Peroxide-Induced Apoptosis by Facilitating Mitochondria-Dependent Pathway in Vascular Smooth Muscle Cells. Br J Pharmacol. [Epub: Ahead of Print]

Zhang Y., Wang X., Wang H., Jiao J., Li Y., Fan E., Zhang L. and Bachert C. (2015). TMEM16A-Mediated Mucin Secretion in IL-13-Induced Nasal Epithelial Cells From Chronic Rhinosinusitis Patients. Allergy Asthma Immunol Res. 7(4):367-75.

Zhang, X., H. Li, H. Zhang, Y. Liu, L. Huo, Z. Jia, Y. Xue, X. Sun, and W. Zhang. (2017). Inhibition of transmembrane member 16A calcium-activated chloride channels by natural flavonoids contributes to flavonoid anticancer effects. Br J Pharmacol 174: 2334-2345.

Zhang, X.D., J.H. Lee, P. Lv, W.C. Chen, H.J. Kim, D. Wei, W. Wang, C.R. Sihn, K.J. Doyle, J.R. Rock, N. Chiamvimonvat, and E.N. Yamoah. (2015). Etiology of distinct membrane excitability in pre- and posthearing auditory neurons relies on activity of Cl- channel TMEM16A. Proc. Natl. Acad. Sci. USA 112: 2575-2580.

Zhao, P., A. Torcaso, A. Mariano, L. Xu, S. Mohsin, L. Zhao, and R. Han. (2014). Anoctamin 6 Regulates C2C12 Myoblast Proliferation. PLoS One 9: e92749.



1.A.17.1 The Anoctamin (ANO) Family


Examples:

TC#NameOrganismal TypeExample
1.A.17.1.1

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 asCaMKIIδ (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 posthearing 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). 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).  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).  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).

Animals

Anoctamin 1a of Homo sapiens (Q5XXA6)

 
1.A.17.1.10

Anoctamin, Anoh-2.  Present in mechanoreceptive neurons and spermatheca (Wang et al. 2013).

Animals

Anoh-2 of Caenorhabditis elegans

 
1.A.17.1.11Anoctamin-like protein At1g73020PlantsAt1g73020 of Arabidopsis thaliana
 
1.A.17.1.12

Ca-ClC Family homologue

Ciliates

Ca-ClC homologue of Paramecium tetraurelia (A0CAP8)

 
1.A.17.1.13

Ciliate CaClC homologue

Alveolata

CaClC homologue of Paramecium tetraurelia (A0CIB0)

 
1.A.17.1.14

Water mold Anoctamin-like protein

Animal

Anoctamin-like protein of Phytophthora infestans (D0NGF4)

 
1.A.17.1.15

Uncharacterized protein

Fungi

Uncharacterized protein of Schizosaccharomyces japonicus

 
1.A.17.1.16

Anoctamin-like protein

Alveolata

Anoctamin-like protein of Oxytricha trifallax

 
1.A.17.1.17

TMEM16 (Ist2) ion channel/phospholipid scramblase (Malvezzi et al. 2013).

Fungi

Ist2 of Aspergillus fumigatus (Neosartorya fumigata)

 
1.A.17.1.18

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).

Fungi

TMEM16 of Nectria haematococca (Fusarium solani subsp. pisi)

 
1.A.17.1.19

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

 
1.A.17.1.2

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.

Metazoa

Anoctamin 1b of Homo sapiens (Q75UR0)

 
1.A.17.1.20

Anoctamin 3, ANO3 or KCNT1, of 981 aas and 9 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).

ANO3 or KCNT1 of Homo sapiens

 
1.A.17.1.21

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).

Ano5 of Homo sapiens

 
1.A.17.1.22

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).

Subdued of Drosophila melanogaster

 
1.A.17.1.23

ANO-like protein of 921 aas and 9 predicted TMSs.

ANO-L family protein of Strongylocentrotus purpuratus (Purple sea urchin)

 
1.A.17.1.24

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

 
1.A.17.1.25

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

 
1.A.17.1.26

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).

TMEM16K of Homo sapiens

 
1.A.17.1.27

Anoctamin 7, ANO7, 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 of Homo sapiens

 
1.A.17.1.28

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

 
1.A.17.1.3

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).

Animals

TMEM16B of Homo sapiens (Q9NQ90)

 
1.A.17.1.4

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 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 transported 4.5 x 104 lipids per second at 25 degrees C, with an activation free energy of 47 kJ/mol, suggestiong a channel-dependent, facilitated diffusion,"stepping-stone" mechanism (Watanabe et al. 2018).

Animals

Anoctamin-6 of Homo sapiens (Q4KMQ2)

 
1.A.17.1.5

Anoctamin-9 (Transmembrane protein 16J) (Tumor protein p53-inducible protein 5) (p53-induced gene 5 protein).  It promotes pancreatic tumorigenesis (Jun et al. 2017).

Animals

ANO9 of Homo sapiens

 
1.A.17.1.6

Uncharacterized protein

Fungi

Uncharacterized protein of Batrachochytrium dendrobatidis

 
1.A.17.1.7

Anoctamin-like protein

Amoebozoa (Slime molds)

amoctamin-like protein of Dictyostelium purpureum

 
1.A.17.1.8

Uncharacterized protein

Stremenopiles

unchacterized protein of Aureococcus anophagefferens

 
1.A.17.1.9

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).

Animals

Anoh-1 of Caenorhabditis elegans

 


1.A.17.2 The Anoctamin-like (ANO-L) Family


Examples:

TC#NameOrganismal TypeExample
1.A.17.2.1

DUF590 family protein

Slime molds

DUF590 protein of Dicyostelium discoideum (Q54BH1)

 
1.A.17.2.2

TMEM16 homologue of 701 aas.

Heterolobosea

TMEM16 homologue of Naegleria gruberi (Amoeba)

 
1.A.17.2.3

Anoctamin homologue of 689 aas

Cryptophyta

Anoctamin of Guillardia theta

 
1.A.17.2.4

DUF590 homologue of 487 aas

Amoebozoa

DUF590 homologue of Entamoeba nuttalli

 
1.A.17.2.5

DUF590 protein of 914 aas

DUF590 protein of Allomyces macrogynus

 
1.A.17.2.6

Uncharacterized protein of 569 aas and 8 predicted TMSs.

UP of Dictyostelium fasciculatum (Slime mold)

 


1.A.17.3 The Calcium-permeable Stress-gated Cation Channel-like 1 (CSC-1) Family


Examples:

TC#NameOrganismal TypeExample
1.A.17.3.1

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).

Stramenopiles

UP of Thalassiosira pseudonana (Marine diatom) (Cyclotella nana)

 
1.A.17.3.10

Uncharacterized protein of 1080 aas

Plants

UP of Ostreococcus lucimarinus

 
1.A.17.3.11

Anoctamin homologue of 1265 aas

Alveolata (ciiates)

Anoctamin homologue of Tetrahymena thermophila

 
1.A.17.3.12

Uncharacterized protein of 995 aas and 8 TMSs.

UP of Tetrahymena thermophila

 
1.A.17.3.13

Uncharacterized protein of 10 TMSs in a 3 + 4 +3 arrangement

UP of Paramecium tetraurelia

 
1.A.17.3.14

Uncharacterized protein of 888 aas and 10 TMSs in a 3 + 4 + 3 arrangement

UP of Paramecium tetraurelia

 
1.A.17.3.15

Uncharacterized protein of 958 aas and 11 or 12 TMSs in a 3 or 4 + 5 +3 arrangement.

UP of Paramecium tetraurelia

 
1.A.17.3.2

Uncharacterized protein of 842 aas and 9 TMSs.

Stramenopiles

UP of Thalassiosira pseudonana (Marine diatom) (Cyclotella nana)

 
1.A.17.3.3

Uncharacterized protein of 835 aas and 9 TMSs.

Stramenopiles

UP of Phytophthora parasitica (Potato buckeye rot agent)

 
1.A.17.3.4

Uncharacterized protein of 1231 aas and 9 TMSs

Stramenopiles

UP of Aureococcus anophagefferens (Harmful bloom alga)

 
1.A.17.3.5

Uncharacterized protein of 945 aas and 8 TMSs

Stramenopiles

UP of Ectocarpus siliculosus (Brown alga)

 
1.A.17.3.6

Uncharacterized protein of 1437 aas

Haptophyceae

UP of Emiliania huxleyi

 
1.A.17.3.7

Uncharacterized protein of 1150 aas

Ichthyosporea

UP of Capsaspora owczarzaki

 
1.A.17.3.8

DUF590/putative methyltransferase of 1221 aas and 10 TMSs.

Alveolata

DUF490 homologue of Oxytricha trifallax

 
1.A.17.3.9

DUF590 homologue of 1026 aas and 10 TMSs

Alveolata

DUF590 homologue of Paramecium tetraurelia (ciliate)

 


1.A.17.4 The Transmembrane Channel (TMC) Family


Examples:

TC#NameOrganismal TypeExample
1.A.17.4.1

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).

Animals

TMC2 of Mus musculus (Q8R4P4)

 
1.A.17.4.10

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).

 

Animal

TMC6 of Homo sapiens

 
1.A.17.4.11

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).

 

Animals

TMC8 of Homo sapiens

 
1.A.17.4.12

Transmembrane channel protein 3, Tmc3 of 1130 aas (Kurima et al. 2003; Beurg et al. 2014).

Animals

Tmc3 of Mus musculus

 
1.A.17.4.13

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).

Animals

Tmc1/Tmc2/Pcdh15 complex of Danio rerio (Zebrafish) (Brachydanio rerio)

 
1.A.17.4.14

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).

TMC4 of Homo sapiens

 
1.A.17.4.15

Tmc1 of 760 aas and 10 TMSs.  96% identical to mouse TMC1 (TC# 1.A.17.4.6).  Vairants responsible for hereditary hearing loss have been identified (Wang et al. 2018). There are varying numbers of channels per mechanoelectrical transduction (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 that dextran permeation requires TMC1/2 proteins and functional MET channels (Ballesteros et al. 2018). TMC1 is a pore-forming component of sensory transduction channels in auditory and vestibular hair cells (Pan et al. 2018).

TMC1 of Homo sapiens

 
1.A.17.4.2

Transmembrane channel-like protein-B, Tmc8 (EVER2).  Occurs in the endoplasmic reticulum where it functions to release Ca2+ and Zn2+ and supresses Cl- currents (Sirianant et al. 2014). 

Animals

Tmc8 of Mus musculus (Q7TN58)

 
1.A.17.4.3

Hypothetical protein, HP

Choanoflagellida

HP of Salpingoeca sp. (F2U2C0)

 
1.A.17.4.4

Hypothetical protein, HP

Ichthyosporea

HP of Capsaspora owczarzaki (E9C7I1)

 
1.A.17.4.5

Transmembrane channel-like protein 7, TMC7

Animals

TMC7 of Acromyrmex echinatior (F4X8H9)

 
1.A.17.4.6

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 beiing the hair cell mechanosensitive channel has been evaluated (Fettiplace 2016).  The human orthologue (UniProt acc # Q8TDI8) is 96% identical.

Animals

Tmc1 of Mus musculus

 
1.A.17.4.7

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).

Animals

Tmc-1 of Caenorhabditis elegans

 
1.A.17.4.8

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).

Animals

Tmc2 of Caenorhabditis elegans

 
1.A.17.4.9

Tmc receptor/channel of 1932 aas. Plays a role in Drosophila proprioception and the sensory control of larval locomotion (Guo et al. 2016).

Animals (fruit flies)

Tmc of Drosophila melanogaster

 


1.A.17.5 The Calcium-permeable Stress-gated Cation Channel (CSC) Family


Examples:

TC#NameOrganismal TypeExample
1.A.17.5.1

Uncharacterized protein, DUF221, of 703 aas

Plants

UP of Zea mays

 
1.A.17.5.10

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).

Plants

CSC1 of Arabidopsis thaliana

 
1.A.17.5.11

Osmotically-gated calcium conductance channel of 782 aas. CSC1 (Hou et al. 2014). Activated under hyperosmotic conditions. There are four paralogues in S. cerevisiae

Fungi

CSC1 of Saccharomyces cerevisiae

 
1.A.17.5.12

The osmosensitive calcium-permeable cation channel, CSC1 of 806 aas.  Activated by hyperosmolarity and Ca2+ (Hou et al. 2014).

Animals

CSC1 of Homo sapiens

 
1.A.17.5.13

Uncharacterized protein of 901 aas

UP of Spironucleus salmonicida

 
1.A.17.5.14

Uncharacterized protein of 1267 aas and 12 TMSs

UP of Dictyostelium discoideum (Slime mold)

 
1.A.17.5.15

Uncharacterized protein of 1548 aas and 12 TMSs.

UP of Thalassiosira pseudonana (Marine diatom) (Cyclotella nana)

 
1.A.17.5.16

Uncharacterized protein of 1172 aas

UP of Phytomonas sp. isolate EM1

 
1.A.17.5.17

Uncharacterized protein of 1258 aas and 11 TMSs.

UP of Agaricus bisporus (White button mushroom)

 
1.A.17.5.18

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

 
1.A.17.5.19

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 helicesforms an anchor in the lipid bilayer and may be essential to osmosensing.

OSCA1.2 of Arabidopsis thaliana (Mouse-ear cress)

 
1.A.17.5.2

Uncharacterized protein of 816 aas containe a DUF221 domain

Animals

UP of Danio rerio

 
1.A.17.5.3

Uncharacterized transmembrane protein 63B of 832 aas with a DUF221 domain.

Animals

UP of Homo sapiens

 
1.A.17.5.4

Uncharacterized transmembrane protein 63B of 832 aas with a DUF221 domain.

Amoebozoa

UP of Acanthamoeba castellanii

 
1.A.17.5.5

Uncharacterized protein of 853 aas with a DUF221 domain.

Fungi

UP of Botryotinia fuckeliana

 
1.A.17.5.6

Phosphate metabolism protein 7, Phm7

Yeast

Phm7 of Saccharomyces cerevisiae

 
1.A.17.5.7

Sporulation-specific protein 75, Spo75

Yeast

Spo75 of Saccharomyce cerevisiae

 
1.A.17.5.8

RSN-1-like protein of 957 aas

Yeast

RSN-1-like protein of Saccharomyces kudriavzevii

 
1.A.17.5.9

Early response to dehydrate stress protein, ERD4 of 785 aas

Plants

ERD4 of Arabidopsis thaliana

 


1.A.17.6 The Transmembrane Channel-like (TMC-L) Family


Examples:

TC#NameOrganismal TypeExample
1.A.17.6.1

Uncharacterized protein of 878 aas and 7 putative TMSs.

Alveolata

UP of Oxytricha trifallax

 
1.A.17.6.10

Uncharacterized protein of 707 aas and 10 TMSs

UP of Plasmodiophora brassicae

 
1.A.17.6.2

TMC-like protein 8 of 890 aas and 8 TMSs

Alveolata

TMC homologue of Oxytricha trifallax

 
1.A.17.6.3

Uncharacterized protein of 834 aas and 7 TMSs

Alveolata

UP of Oxytricha trifallax

 
1.A.17.6.4

Uncharacterized protein of 912 aas and 10 TMSs

Stramenopiles

UP of Phytophthora parasitica (Potato buckeye rot agent)

 
1.A.17.6.5

Uncharacterized protein of 620 aas and 9 TMSs

Stremenopiles

UP of Ectocarpus siliculosus (Brown alga)

 
1.A.17.6.6

Uncharacterized protein of 865 aas and 10 TMSs

Cryptophyta

UP of Guillardia theta

 
1.A.17.6.7

TMC protein of 890 aas and 10 TMSs

Alveolata

TMC protein of Tetrahymena thermophila

 
1.A.17.6.8

Uncharacterized protein of 1057 aas and 10 TMSs.

UP of Tetrahymena thermophila

 
1.A.17.6.9

Uncharacterized protein of 867 aas and 10 TMSs.

UP of Saprolegnia diclina

 


1.A.17.7 The Calcium-permeable Stress-gated Cation Channel-like 2 (CSC-L2) Family


Examples:

TC#NameOrganismal TypeExample
1.A.17.7.1

Uncharacterized protein of 836 aas and 12 TMSs.

UP of Giardia intestinalis (Giardia lamblia)

 
1.A.17.7.2

Uncharacterized protein of 637 aas and 8 TMSs.

UP of Spironucleus salmonicida

 
1.A.17.7.3

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

 
1.A.17.7.4

Uncharacterized Anoctamin homologue of 502 aas and 8 putative TMSs

UP of Spironucleus salmonicida

 
1.A.17.7.5

Uncharacterized anoctamin homologue of 823 aas and 8 predicted TMSs in a 3 + 2 + 3 arrangement.

UP of Giardia intestinalis (Giardia lamblia)

 
Examples:

TC#NameOrganismal TypeExample