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)