2.A.49 The Chloride Carrier/Channel (ClC) Family
The ClC family is a large family consisting of thousands of sequenced proteins derived from Gram-negative and Gram-positive bacteria, archaea, and all kinds of eukaryotes. These proteins are essentially ubiquitous, although they are not encoded within the genomes of several prokaryotes with small genomes. Sequenced proteins vary in size from 395 amino acyl residues (M. jannaschii) to 988 residues (humans). Many organisms contain multiple ClC family paralogues. For example, E. coli and Synechocystis both have two paralogues; mammals have nine paralogues, and C. elegans has at least five. Of the nine known members in mammals, mutations in three of the corresponding genes cause human diseases (Matulef and Maduke, 2007). MstE (1.A.26.1.2), CLC (2.A.49.6.1) and HlyC/CorC (HCC; 9.A.40.1.2) may all share a hydrophilic domain, and not all members of 9.A.40 have a transmembrane region. ClC family members may be able to transport a variety of anions inclluding nitrate (Kumar et al. 2022). CLCs are involved in cell growth, proliferation, and cell cycle, and they may be involved in prostate cancer (PCa). Silencing CLCN2 or CLCN6 exerts inhibitory effects on energy metabolism in PCa (Luo et al. 2022).
The ClC transport protein family consists of H+-gated Cl- channels, H+/Cl- antiporters and H+/NO3- antiporters. The pore is obstructed at its external opening by a glutamate side-chain which acts as a gate for Cl- passage in channels and as the H+ binding site for H+ exchange. The activity of ClC-2, a genuine Cl- channel, has a biphasic response to extracellular pH with activation by moderate acidification followed by abrupt channel closure at pH values lower than 7. A sensor couples extracellular acidification to closure of the channel, an extracellularly-facing histidine (His5320 at the N-terminus of transmembrane helix Q (Niemeyer et al. 2009). Neutralization leads to channel closure in a cooperative manner. Acidification-dependent activation of ClC-2 is voltage-dependent and probably mediated by protonation of pore gate glutamate 207. Intracellular Cl- acts as a voltage-independent modulator, regulating the pKa of the protonatable residue. Voltage dependence of ClC-2 occurs by hyperpolarization-dependent penetration of protons from the extracellular side to neutralize the glutamate gate deep within the channel, allowing Cl- efflux. This is reminiscent of a partial exchanger cycle, suggesting that the ClC-2 channel evolved from its transporter counterparts (Niemeyer et al. 2009).
The nine mammalian ClC isoforms differ in tissue distribution and subcellular localization. Some of these are plasma membrane Cl- channels, which play important roles in transepithelial transport and in dampening muscle excitability. Other ClC proteins localize mainly to the endosomal-lysosomal system where they may facilitate luminal acidification or regulate luminal chloride concentration. All vesicular ClCs may be Cl-/H+-exchangers, as shown for the endosomal ClC-4, -5 and -7 proteins. Human diseases include myotonia, renal salt wasting, kidney stones, deafness, blindness, male infertility, leukodystrophy, osteopetrosis, lysosomal storage disease and defective endocytosis (Jentsch, 2008). CLC channels display two different types of 'gates,' 'protopore' gates that open and close the two pores of a CLC dimer independently of each other, and common gates that act on both pores simultaneously (Ludwig et al. 2013). The chloride and proton pathways have been identified and proposed, the latter involving a 'water wire' (Han et al. 2013). The CLC superfamily genes in tea plants (Camellia sinensis) have been identifed and examined (Xing et al. 2020).
Two gating mechanisms control the opening and closing of Cl- channels in this family: fast gating, which regulates opening and closing of the individual pores in each subunit of the dimeric transporter, and slow (or common) gating, which simultaneously controls gating of both subunits. Yu et al. 2015 found that intracellularly applied Cd2+ reduces the current of CLC-0 because of its inhibition on the slow gating. They identified CLC-0 residues C229 and H231, located at the intracellular end of the transmembrane domain near the dimer interface, as the Cd2+-coordinating residues. Inhibition of the current of CLC-0 by Cd2+ was enhanced by mutation of I225W and V490W at the dimer interface.
Methanococcus jannaschii and Saccharomyces cerevisiae only have one ClC family member each. With the exception of the large Synechocystis paralogue, bacterial proteins are usually small (395-492 residues) while eukaryotic proteins are usually larger (687-988 residues). These proteins exhibit 12 putative transmembrane α-helical spanners (TMSs) and appear to be present in the membrane as homodimers. A 12 TMS topology with the N- and C-termini in the cytoplasm was suggested.
The structure of the E. coli EriC (TC #2.A.49.5.1) ClC family member has been reported at 3.0 Å resolution (Dutzler et al., 2002; Mindell et al., 2001). Two identical water-filled pores, each within a single subunit of the dimeric channel complex were revealed. Each subunit consists of two roughly repeated halves that span the membrane with 5 TMSs each and opposite orientations in the membrane. This antiparallel architecture defines a selectivity filter in which Cl- is stabilized by electrostatic interactions with α-helix dipoles and chemical coordination with nitrogen and hydroxyl groups in the protein. This protein has been shown to mediate the extreme acid resistance response (Iyer et al., 2002). Thus, E. coli is proposed to use either one of its two ClC channels as electrical shunts for an outwardly directed virtual pump that is linked to amino acid decarboxylation (Iyer et al., 2002). The crystal structure of a eukaryotic CLC transporter defines an intermediate state in the transport cycle (Feng et al., 2010).
The E. coli EriC (also called ClC-ecl) has been studied leading to the conclusion that it is not a simple channel, but instead catalyzes Cl-:H+ antiport with a stoichiometry of 2.1. They can thus function as carriers (Accardi and Miller, 2004). The authors note that the eukaryotic ClC-0, ClC-1 and ClC-2 are unambiguously Cl--selective channels which display proton-dependent gating but show no indication of H+ permeability. Moreover, they note that the 3-D structure published by Dutzler et al. (2002, 2003) does not actually show a transmembrane pore. A conserved glutamate, when mutated in ClC-0 or ClC-1 eliminates the normal pH-dependency of the Cl- flux, while in EriC, this glutamate, E148, may provide the pathway for the proton. E148A or E148Q mutants do not transport H+ but do transport Cl- in an uncoupled process.
Kieseritzky and Knapp (2011) modeled four stable buried waters into both subunits of the wild type E.coli CIC channel (EClC). They form a 'water wire' connecting Glu-203 with the chloride at the central site, which in turn connects to Glu-148, the hypothetical proton exit site. Assuming the transient production of hydrochloride in the central chloride binding site of EClC, the water wire could establish a transmembrane proton transport pathway starting from Glu-203 all the way downstream onto Glu-148. EClC evolves through states involving up to two excess protons and between one and three chlorides, which fulfill the experimentally observed 2:1 stoichiometry. Y445F and E203H mutants of EClC can operate similarly, thus explaining why they exhibit almost WT activity. The proposed mechanism thus involves coupled chloride-proton transport (Kieseritzky and Knapp, 2011).
Mutating a 'gating glutamate' (Glu-224 in ClC-4 and Glu-211 in ClC-5) converted these exchangers into anion conductances, as did the neutralization of another, intracellular 'proton glutamate' in ecClC-1 (Zdebik et al., 2007). Neutralizing the proton glutamate of ClC-4 (Glu-281) and ClC-5 (Glu-268) abolished Cl- and H+ transport although surface expression was unchanged. Uncoupled Cl- transport could be restored in the ClC-4 (E281A) and ClC-5 (E268A) proton glutamate mutants by neutralizing the gating glutamates, suggesting that wild type proteins transport anions only when protons are supplied through a cytoplasmic H+ donor. Each monomeric unit of the dimeric protein is able to carry out Cl-/H+ exchange independently of the activity of the neighboring subunit. NO3- or SCN- transport is partially uncoupled from H+ countertransport but still depends on the proton glutamate. Thus, Cl-/H+ exchange catalyzed by the endosomal ClC-4 and -5 proteins apparently relies on proton delivery from an intracellular titratable residue at position 268 (ClC-5).
Picollo and Pusch (2005) and Scheel et al. (2005) have shown that endosomal ClC4 and ClC5 (both human disease proteins) are electrogenic Cl-/H+ antiporters, probably exhibiting a 2:1 stoichiometry. They showed that mutation of the conserved glutamate, aligning with that in EriC of E. coli abolished proton (but not chloride) transport. They confirmed that ClC-0, ClC-2, ClC-Ka function like channels. Thus, a single residue can convert a channel to a carrier. It seems that point mutations can create ‘broken channels’ out of these carriers. Because Cl-:H+ antiporters function with subunit cross-linking, large quaternary rearrangements, such as those known to occur for 'common gating' in ClC channels, are probably not necessary for the ion transport cycle (Nguitragool and Miller, 2007).
All eukaryotic ClC channels so far examined contain two C-terminal CBS domains, each of 50 residue. CBS domains are found in various globular proteins. The Torpedo ClC-0 and the E. coli YadQ have been reported to have two channels and function by a double barrelled mechanism, one per subunit. Some evidence suggests that for ClC-0, ClC-1 and ClC-2, each subunit bears a single channel, and the association of the subunits of these channel proteins to form homo- or heterodimers does not alter their conductance properties.
The CLC family is formed by two, not so distinct, sub-classes of membrane transport proteins: Cl- channels and H+/Cl- exchangers (Accardi and Picollo, 2010). All CLC's are homodimers with each monomer forming an individual Cl- permeation pathway which appears to be largely unaltered in the two CLC sub-classes. Key residues for ion binding and selectivity are also highly conserved. Most CLC's have large cytosolic carboxy-terminal domains containing two cystathionine beta-synthetase (CBS) domains. The C-termini are critical regulators of protein trafficking and directly modulate Cl- by binding intracellular ATP, H+ or oxidizing compounds.
All functionally characterized members of the ClC family transport chloride, some in a voltage-regulated process. These channels serve a variety physiological functions (cell volume regulation; membrane potential stabilization; signal transduction; transepithelial transport, etc.). Different homologues in humans exhibit differing anion selectivities, i.e., ClC-4 and ClC-5 share a NO3- > Cl- > Br- > I- conductance sequence, while ClC-3 has an I- > Cl- selectivity.
The ClC-4 and ClC-5 channels/carriers and others exhibit outward rectifying currents with currents only at voltages more positive than +20mV. Some but not other ClC channels are permeable to the low conductance blockers, I- and SCN-. ClCα-1 has been studied in detail. ClO4- and SCN- are more permeant than Br-, NO3- or ClO3-, and the hydrophobic anions, benzoate and hexanoates, are more permeable than smaller anions such as BrO3-. It is clear that ClCs are not specific for chloride, but are general anion channels and carriers (Miller, 2006).
A genetic defect of ClC-5 in humans is the cause of Dent's disease. This protein is expressed in endosomes of the proximal tubule. Disruption of the corresponding clcn5 gene in mice causes proteinuria by reducing apical proximal tubular endocytosis. This delays internalization of the apical transporters NaPi-2 and NHE3 (Piwon et al., 2000). It has been suggested that it plays a role in acidification of both endocytic and exocytic vesicles involved in protein trafficking.
Plants need nitrate for growth and store most of it in the central vacuole. Some members of the ClC family, such as the torpedo-fish ClC-0 and mammalian ClC-1, are anion channels, whereas the E. coli EriC and mammalian ClC-4 and ClC-5 are Cl-/H+ exchangers. Some plant members of the ClC family may be anion channels involved in nitrate homeostasis. However, Arabidopsis thaliana ClCa is localized to the tonoplast membrane of the plant vacuole. De Angeli et al. (2006) have demonstrated that ClCa is able to accumulate nitrate in the vacuole and behaves as a NO3-/H+ exchanger.
Feng et al. 2010 have determined the structure of a eukaryotic CLC transporter from the red alga (Rhodophyta) at 2.5 angstrom resolution. Cytoplasmic cystathionine beta-synthase (CBS) domains are strategically positioned to regulate the ion-transport pathway, and many disease-causing mutations in human CLCs reside on the CBS-transmembrane interface. Comparison with prokaryotic CLC shows that a gating glutamate residue changes conformation and suggests a basis for 2:1 Cl-/H+ exchange and a simple mechanistic connection between CLC channels and transporters.
Six ClC-type chloride channel genes have been identified in Caenorhabditis elegans, termed clh-1 through clh-6, but clh-2, clh-3, and clh-4 may code for multiple channel variants (Nehrke et al. 2000). CLH-5 is expressed ubiquitously, CLH-6 is expressed mainly in nonneuronal cells, and the remaining isoforms vary from those restricted to a single cell to those expressed in over a dozen cells of the nematode. Both CLH-1 and CLH-3b produced strong, inward-rectifying chloride currents similar to those arising from mammalian ClC2, but which operate over different voltage ranges.
In eukaryotes, ClC proteins play a role in the stabilization of membrane potential, epithelial ion transport, hippocampal neuroprotection, cardiac pacemaker activity and vesicular acidification. Moreover, mutations in the genes encoding ClC proteins can cause genetic disease in humans (Abeyrathne et al. 2016). In prokaryotes, the Cl-/H+ antiporters, such as ClC-ec1 found in Escherichia coli promote proton expulsion in the extreme acid-resistance response common to enteric bacteria. Structural features include a complicated transmembrane topology with 18 α-helices in each subunit and an anion-coordinating region in each subunit. Several different approaches such as X-ray crystallography, NMR, biochemical studies, and molecular dynamics simulations have been applied to the study of ClC proteins (Abeyrathne et al. 2016).
ClC proteins mediate the movement of Cl- across the membrane. In eukaryotes, ClC proteins play roles in membrane potential stabilization, epithelial ion transport, hippocampal neuroprotection, cardiac pacemaker activity and vesicular acidification, and mutations in the genes encoding ClC proteins can cause genetic disease in humans (Abeyrathne et al. 2016). In prokaryotes, the Cl-/H+ antiporters, such as ClC-ec1 found in Escherichia coli promote proton expulsion in the extreme acid-resistance response common to enteric bacteria. Structural and functional studies of the prokaryotic protein have revealed unique structural features, including a complicated transmembrane topology with 18 alpha-helices in each subunit with a central anion-coordinating region. Several different approaches such as X-ray crystallography, NMR, biochemical studies, and molecular dynamics simulations have been applied to the study of ClC proteins.
ClCs are expressed in both plasma and intracellular membranes of cells from almost all eukaryotic organisms. ClC proteins form transmembrane dimers, in which each monomer displays independent ion conductance. Eukaryotic members possess a large cytoplasmic domain containing two CBS domains involved in transport modulation. ClC proteins function as either Cl- channels or Cl-/H+ exchangers, although all ClC proteins probably share the same basic architecture (Poroca et al. 2017). ClC channels have two gating mechanisms: a relatively well-studied fast gating mechanism, and a poorly studied slow gating mechanism. ClCs are involved in a wide range of physiological processes, including regulation of the resting membrane potential in skeletal muscle, facilitation of transepithelial Cl- reabsorption in kidneys, and control of pH and Cl- concentration in intracellular compartments through coupled Cl-/H+ exchange mechanisms. Several inherited diseases result from C1C gene mutations, including myotonia congenita, Bartter's syndrome (types 3 and 4), Dent's disease, osteopetrosis, retinal degeneration, and lysosomal storage diseases (Poroca et al. 2017).
CLC anion transporters are found in all phyla and form a gene family of eight members in mammals. Two CLC proteins, each of which completely contains an ion translocation parthway, assemble to homo- or heteromeric dimers that sometimes require accessory β-subunits for function (Jentsch and Pusch 2018). CLC proteins can be anion channels or anion/proton exchangers. Structures of these two CLC protein classes are surprisingly similar. Extensive structure-function analyses have identified residues involved in ion permeation, anion-proton coupling and gating. In mammals, ClC-1, -2, -Ka/-Kb are plasma membrane Cl- channels, whereas ClC-3 through ClC-7 are 2Cl-/H+-exchangers in endolysosomal membranes. Biological roles of CLCs were mostly studied in mammals, but also in plants and model organisms like yeast and Caenorhabditis elegans. CLC Cl- channels have roles in the control of electrical excitability, extra- and intracellular ion homeostasis, and transepithelial transport, whereas anion/proton exchangers influence vesicular ion composition and impinge on endocytosis and lysosomal function. The surprisingly diverse roles of CLCs are highlighted by human and mouse disorders elicited by mutations in their genes. These pathologies include neurodegeneration, leukodystrophy, mental retardation, deafness, blindness, myotonia, hyperaldosteronism, renal salt loss, proteinuria, kidney stones, male infertility, and osteopetrosis. Jentsch and Pusch 2018 reviewed all aspects of CLC proteins with emphasis on biophysical structure-function analyses and on the cell biological and organismal roles of mammalian CLCs in disease. They can be chloride conducting channels and proton-chloride antiporters. All members in this family consist of two identical protein subunits, and each subunit forms an independent ion-transport pathway, a structural architecture known as 'double barrel.' These CLC proteins serve biological functions ranging from membrane excitability and cell volume regulation to acidification of endosomes (Kwon et al. 2022).
Note: The ClC family was previously given the TC# 1.A.11.
The generalized transport reaction catalyzed by carriers of the ClC family is:
2 Anions (in) + H+ (out) ⇌ 2 Anions (out) + H+ (in).
The generalized transport reaction catalyzed by channels of the ClC family is:
Anion (in) ⇌ Anion (out)
Chloride channel protein of 2075 aas and 10 TMSs.
CLC family member of Toxoplasma gondii
Voltage and (possibly) ATP-gated Cl- channel, ClC-1 or CLCN1 (Bennetts et al., 2005; Zifarelli and Pusch, 2008). It may also transport other anions such as thiocyanate, perchlorate, bromide, nitrate, chlorate and iodide. When mutant, it causes dominant and recessive myotonia. Myotonia congenita is a genetic disease characterized by impaired muscle relaxation after forceful contraction (Orsini et al. 2020). It has a large cytoplasmic C-terminal domain bearing two CBS (cystathionine-β-synthase) domains. Alternative splicing is a posttranscriptional mechanism regulating chloride conductance during muscle development (Lueck et al. 2007). ClC-1-like channels are preferentially located at the somata of medium spiny neurons and can modulate neuronal excitability (Yarotskyy et al. 2022).
ClC1 or CLCN1 of Homo sapiens (P35523)
Chloride transporter/chloride channel (ClC) family protein of 1604 aas (Docampo et al. 2013).
CLC family member of Toxoplasma gondii
The voltage-gated torpedo chloride channel, CLC-0, of 810 aas. This channel is thought to ensure the high conductance of the non-innervated membrane of the electrocyte necessary for efficient current generation caused by sodium influx through the acetylcholine receptor at the innervated membrane. ClC-0 is a homodimer with separate pores in each subunit. Each protopore can be opened and closed independently from the other pore by a ""fast gate"". A common, slow gate acts on both pores simultaneously. The opening of the fast gate is controlled by a critical glutamate (E166), whose protonation state determines the fast gate's pH dependence (Zifarelli and Pusch 2010). Extracellular protons likely arrive directly at E166, but protonation of E166 from the inside may be mediated by the dissociation of an intrapore water molecule. The OH- anion resulting from dissociation of water is stabilized in one of the anion binding sites of the channel, competing with intracellular Cl-. Proton translocation drives irreversible gating transitions associated with the slow gate (Zifarelli and Pusch 2010). The structure of the cytoplasmic domain has been solved (Meyer and Dutzler 2006). It contains a folded core of two tightly interacting cystathionine beta-synthetase (CBS) subdomains which are connected by a 96 residue mobile linker. The domains form dimers, thereby extending the 2-fold symmetry of the transmembrane pore.
CLC-0 of Torpedo californica (Pacific electric ray)
Chloride (bromide; iodide) channel-2, CLC-2, CLC2, CLCN2; 94% identical to the rat orthologue, 2.A.49.2.6. Hyperpolarization activates CLC-2 mainly by driving intracellular anions into the channel pores. Protonation by extracellular H+ plays a minor role in dislodging the glutamate gate (De Jesús-Pérez et al. 2016). The absence of ClC-2 results in less differentiated colonic crypts and increased tumorigenicity associated with colitis via dysregulation (Jin et al. 2018).
CLC-2 of Homo sapiens
Clh-3; Clc-3, Clh3, Clc3 of 1001 aas and 11 TMSs. Isoform a: Voltage-gated chloride channel (Rutledge et al. 2002; Denton et al. 2005). Insensitive to depolarizing voltages; requires low voltages for activation; insensitive to chloride levels but has a mild sensitivity to low pH. Channel gating properties are conferred by the cytoplasmic C-terminus (He et al. 2006). Plays a role in egg laying by modulating hermaphrodite-specific neuron excitability and the ovulatory contractions of gap-junction-coupled gonadal sheath cells (Branicky et al. 2014). When active, may prevent tubular formation of the excretory canals (Hisamoto et al. 2008). Activated during oocyte meiotic maturation and by membrane hyperpolarization and cell swelling (Denton et al. 2004). Inhibited by Zn2+ and to a lesser extent by Cd2+(Rutledge et al. 2001). Isoform b: Voltage-gated chloride channel sensitive to depolarizing conditioning voltages; requires stronger voltages for activation and activation is slower, is inhibited by low concentrations of chloride and is activated by low pH. Channel gating properties are conferred by the cytoplasmic C-terminus (He et al. 2006).
Clh3 of Caenorhabditis elegans
The voltage-gated chloride channel, CLCNKB, CLCCK or CLC-Kb of 687 aas and 10 TMSs in a 2 + 2 + 2 + 2 + 2 arrangement. These anion channels have several functions including the regulation of cell volume; membrane potential stabilization, signal transduction and transepithelial transport. It may also be important in urinary concentrating mechanisms (Estévez et al. 2001). Inactivated variants in the CLCNKB gene, encoding the basolateral chloride channel ClC-Kb cause classic Bartter syndrome, characterized by hypokalemic metabolic alkalosis and hyperreninemic hyperaldosteronism (Wang et al. 2018).
CLCNKb of Pseudomonas sp. UK4
Chloride channel protein, CLCNKB or ClC-Kb or CLCKB of 687 aas and 10 TMSs in a 2 + 2 + 2 + 2 + 2 TMS arrangement. Voltage-gated chloride channel. Chloride channels have several functions including the regulation of cell volume; membrane potential stabilization, signal transduction and transepithelial transport. May be important in urinary concentrating mechanisms (Estévez et al. 2001).
CLCNKB of Homo sapiens
H+/Cl- exchange transporter 5, ClC-5 (CLCN5; CLCK2) of 816 aas and 12 TMSs in a 1 + 1 + 2 + 2 + 2 + 2 + 2 TMS arrangement. It functions as antiport system and exchanges chloride ions against protons (Neagoe et al. 2010). ClC-5 is expressed in the apical membrane of cyst epithelia and is a likely candidate mediating Cl- secretion into the kidney cyst lumen in the tuberous sclerosis complex (TSC) (Barone et al. 2022).
CLC5 of Homo sapiens
Intracellular (endosomal) outward rectifying kidney Cl-:H (2:1) antiporter ClC5; CLC-5; CLCN5 (nitrate > Cl- = Br- > I- > acetate > gluconate) (Picollo and Pusch, 2005; Scheel et al., 2005); responsible for Dent''s disease in humans, an X-linked disorder characterized by low molecular weight proteinuria, hypercalciuria, and nephrolithiasis (Stechman et al., 2007; Zifarelli and Pusch, 2009; Ashida et al. 2012). Plays a role by facilitating endosomal acidification through neutralization of proton pump currents (Novarino et al., 2010; Rickheit et al., 2010). The carrier is regulated by protons as studied by Picollo et al., (2010). CLC-5 and KIF3B interact to facilitate CLC-5 plasma membrane expression, endocytosis, and microtubular transport (Reed et al., 2010). A point mutations can convert NHE-6 into a nitrate (NO3-):H+ antiporter (Zifarelli and Pusch 2009).
ClC5 of Sus scrofa (Q9GKE7)
The hepatocyte canalicular chloride channel/carrier, CLC-3 or CLCN3 (I- > Br- > Cl-); outward rectifying; inactivated at positive voltages (Shimada et al., 2000). Oxidation induces surface expression of ClC-3 and activation of ClC-3-dependent anion permeability (Kasinathan et al., 2007). It is swell-activated in human neutrophils during a respiratory burst (Ahluwalia, 2008). The ClC-3 Cl-/H+ antiporter becomes uncoupled at low extracellular pH (Matsuda et al., 2010). It transports superoxide, O2- (Hawkins et al. 2007; Fisher 2009). ClC-3 might be involved in modulating vascular remodeling in hypertension and arteriosclerosis (Guan et al. 2006). ClC-3 upregulation may protect against oxidative stress-induced necrosis (Remillard and Yuan 2005).
CLC3 of Rattus norvegicus (P51792)
The kidney medulla voltage-gated dimeric chloride channel, ClC-K1 or CLCNKA. Transports Br-, I- and NO3- as well as Cl-. Regulated by dehydration; functions in the regulation of cell volume, membrane potential stabilization, signal transduction and transepithelial transport. Possibly required for urinary concentration in the kidney (Uchida et al., 1993). The human orthologue when mutated can give rise to salt loss associated with Bartter''s syndrome. In the inner ear, it plays a role in hearing. Activators and inhibitors that may have theraputic benefits are known (Gradogna and Pusch 2010). Regulated by the accessory subunit, Barttin (Wojciechowski et al. 2015).
ClC-K1 of Rattus norvegicus
The kidney thick ascending limb of Henle's loop and collecting duct chloride channel/carrier, ClC-K2 (Br- > I- > Cl- >> cyclamate) (Adachi et al., 1994). Regulated by the accessory subunit, Barttin (Wojciechowski et al. 2015).
ClC-K2 of Rattus norvegicus
The ClC-2 or CLCN2 chloride channel is expressed in many tissues (Cl- ≥ Br- > I-) (Thiemann et al., 1992). Regulated in the retina by Cereblon (Q96SW2) (Hohberger and Enz, 2009). A toxin, GaTx2, is a potent and specific inhibitor of ClC-2, binding with a Ki of 20pM (Thompson et al., 2009). ClC-2 channels constitute part of the background conductance and assist chloride extrusion (Rinke et al., 2010). ClC-2 channels regulate neuronal excitability,vision and fertility, but not intracellular chloride levels (Ratté and Prescott, 2011; Sánchez-Rodríguez et al. 2012). Sequential interactions of chloride and proton ions with the fast gate steer voltage-dependent gating (Sánchez-Rodríguez et al. 2012). The detailed mechanism of proton gating of chloride currents has been presented (Niemeyer et al. 2009).
ClC-2 of Rattus norvegicus
The ClC-A of the trophozoite plasma membrane (Cl->Br->I->F->NO3- ). Functions as an ion channel, not a Cl:H+ antiporter; regulates cell pH and volume (Salas-Casas et al., 2006)
ClC-A of Entamoeba histolytica (Q70S43)
ClC-4 or CLCN4 (catalyzes 2 Cl-:H+ exchange, but NO3- uniport (Bergsdorf et al., 2009). SCN- uncouples Cl- from H+ transport so anions are transported with high unitary transport rates. Voltage then influences transport as is observed for the gating of ion channels (Orhan et al., 2011). Also transports bromide and iodide.
ClC-4 of Homo sapiens (P51793)
The CIC carrier (stoichiometry H+:Cl- = 2:1). 3D structure known to 3.5 Å resolution (PDB# 3ORG; Feng et al., 2010).
CIC carrier of Cyanidioschyzon melolae (PDB 3ORG_A)
NO3-:H+ (or less efficiently, Cl-:H+) antiporter, ClC-A or CLCa (De Angeli et al., 2006; Bergsdorf et al., 2009). Responsible for nitrate uptake into vacuoles (Zifarelli and Pusch, 2009b). It is up-regulated under salt stress conditions (Yang et al. 2018) and may play a role in nitrate and Cd2+ resistance (Liao et al. 2019). The cryoEM structure has been solved at 2.8 Å resolution. An almost identical sequence has Uniprot acc # P92941 and differs from the one included in TCDB between residues 380 and 430, possibly due to sequencing errors. The central nitrate is shifted by approximately 1.4 Å from chloride, which is likely caused by a weaker interaction between the anion and Pro160; the side chains of aromatic residues around the central binding site are rearranged to accommodate the larger nitrate (He et al. 2022).
ClC-A of Arabidopsis thaliana (Q96325)
Mitochondrial inner membrane anion channel (IMAC). Fatty acids induce chloride permeation in rat liver mitochondria probably by activation of the inner membrane anion channel (IMAC) (Schönfeld et al. 2004).
ClC-Nt2 (ClC-1) of Nicotiana tabacum (Q9XF71)
CLC-7, CLC7 or Clcn7 Cl-:H+ antiporter; provides the primary Cl- permeation pathway in the lysosome (Graves et al., 2008). Cl- provides the counter ion for acidification of the lysosomal lumen via the V-type ATPase (Mindell, 2012). It is a slowly voltage-gated 2Cl-/1H+-exchanger and requires Ostm1 for transport activity (Leisle et al., 2011). Common gating (involving both of the two subunits) underlies the slow voltage activation dependent on the C-terminal CBS domain (Ludwig et al. 2013). Lethal CLCN7-related osteopetrosis has been described (Rössler et al. 2021).
CLC-7 of Homo sapiens (P51798)
CLC-6 or CLCN6, Cl-:H+ late endosomal/lysosomal antiporter (43% identical to CLC-7) and 99% identical to the human ortholog (P51797). Cl- > I-; mutating the gating glutamate yielded ohmic anion conductance (Neagoe et al., 2010). A recurrent gain-of-function mutation in CLCN6, encoding the ClC-6 Cl-/H+-exchanger, causes early-onset neurodegeneration (Polovitskaya et al. 2020). Loss of ClC-6 function reduces Golgi calcium stores, which may play a role in vascular contraction and relaxation signaling in vascular smooth muscle cells. Thus, ClC-6 may modulate blood pressure by regulating Golgi calcium reserves, which in turn contribute to vascular smooth muscle function (Klemens et al. 2021).
CLC-6 of Mus musculus (O35454)
Anion transporter with specificity for nitrate (NO3-) and chloride (Cl-), CLCa1, of 811 aas and probably 12 TMSs in a 4 + 4 + 4 TMS arrangement (Nedelyaeva et al. 2019). It is expressed in the roots, and induced with nitrate depletion.
CLCa1 of Suaeda altissima
Cl-:H+ (2:1) antiporter, EriC or ClcA (ClC-ecl) (Accardi and Miller, 2004). The x-ray structure has been determined (PDB 1OTS) Dutzler et al., 2002, 2003). The exchange mechanism involves a conformational cycle of alternating exposure of Cl- and H+ binding sites of both ClC pores to the two sides of the membrane (Miloshevsky et al., 2010). Specific aspects of these conformational changes have been proposed (Wang et al. 2018). Although the protein is present as a homodimer, a single ClC subunit alone is the basic functional unit for transport, and cross-subunit interaction is not required for Cl-/H+ exchange in ClC transporters (Robertson et al., 2010). Glu202 is essential for H+ symport and is on the H+ pathway (Lim et al. 2012). CLC exchangers have two gates that are coupled through conformational rearrangements outside the ion pathway (Basilio et al. 2014). The rotation of E148 plays a critical role in defining the Cl-/H+ coupling (Lee et al. 2016). The channel shows conserved re-entrant helix-coil-helix domains (Tsirigos et al. 2017). Proton transport is intrinsically coupled to protein cavity hydration changes and is influenced by the protein environment (Wang et al. 2018). EriC also transports fluoride (F-) (Baker et al. 2012). Non-polar membrane embedded side-chains in CLC-ec1 play a role in defining dimer stability, but the stoichiometry is contextual to the solvent environment, and L194 is a molecular hot-spot for defining dimerization (Mersch et al. 2021).
EriC of Escherichia coli (P37019)
Slow CLC Cl-:H+ antiporter. 3-d structures known at 3.1 Å resolution (3Q17A) (turnover rate: 20/sec; Cl-:H+ stoichiometry = 2:1). The turnover rate of the E. coli EriC is ~2000/sec. (Jayaram et al., 2011).
Slow CLC of Synechocystis sp. PCC6803 (Q55858)
Voltage-gated ClC-type chloride channel ClcB or MriT of 418 aas.
ClcB of Escherichia coli O6:H1
Voltage-gated ClC-type Cl- channel, ClcB (Phillips et al. 2012).
ClcB of Citrorbacter koseri
H+/Cl- antiporter, ClcA (stoichiometry 1:2) (Phillips et al. 2012).
ClcA of Citrobacter koseri
Voltage-gated Cl- channel, ClC chloride channel core, CCC
CCC of Variovorax paradoxus (C5D215)
Chloride channel protein CLC-e (AtCLC-e) (CBS domain-containing protein CBSCLC3). Present in the thylacoid membrane of the chloroplast. Initially proposed to function in photosynthesis, and later in nitrate assimilation (Herdean et al. 2016).
CLC-E of Arabidopsis thaliana
Putative chloride:H+ antiporter, voltage gated, of 792 aas and 15 TMSs with an N-terminal 4 TMS domain of unknown function found only in a few archaea and a C-terminal CBS domain.
CLC protein of Halobacterium salinarum (Halobacterium halobium)
Uncharacterized membrane protein of 481 aas and 10 - 13 TMSs. This protein was probably incorrectly assigned the function as the membrane constituent of an ABC Zn2+ uptake transporter (Mandal et al. 2019). However, this is unlikely; it is probably a Cl- transporter in agreement with its assignment to TC family 2.A.49.
UP of Thermus thermophilus
Voltage-gated Cl- channel, ClC (YfeO)
YfeO of E. coli (H5KWQ5)
Voltage-gated Cl- channel, ClC (EriC)
EriC of Saccharomonospora azurea (H8G8F4)
Voltage-gated Cl- channel, ClC SCO5964
SCO5964 of Streptomyces coelicolor (O54193)
EriC of Methanoculleus marisnigri (A3CXC7)
Putative chloride channel of 469 aas and 11 putative TMSs
Chloride channel of Galdieria sulphuraria
Fluoride:proton antiporter of 459 aas and 10 TMSs, CLCF, with high selectivity for fluoride over chloride (Brammer et al. 2014). These proteins normally exist as dimers, but the monomers are fully active (Last and Miller 2015).
CLCF of Pirellula staleyi
Fluoride:proton antiporter, CLCF, of 426 aas and 10 TMSs, with specificity for fluoride over other anions (Brammer et al. 2014).
CLCF of Ralstonia pickettii
Fluoride:proton antiporter of 406 aas and 10 TMSs, CLCF (Brammer et al. 2014).
CLCF of Enterococcus casseliflavus
Putative fluoride:proton antiporter of 403 aas and 10 TMSs (Brammer et al. 2014).
CLCF of Lactococcus lactis subsp. cremoris (Streptococcus cremoris)
Anion transporter, EriC, of 452 aas and 10 TMSs. Transports Cl- and F-, and probably other anions (Baker et al. 2012).
EriC of Pseudomonas syringae