Intracellular Proton Access in a Cl?/H+ Antiporter

Chloride-transporting membrane proteins of the CLC family appear in two distinct mechanistic flavors: H⁺-gated Cl⁻ channels and Cl⁻/H⁺ antiporters. Transmembrane H⁺ movement is an essential feature of both types of CLC. X-ray crystal structures of CLC antiporters show the Cl⁻ ion pathway through these proteins, but the H⁺ pathway is known only inferentially by two conserved glutamate residues that act as way-stations for H⁺ in its path through the protein. The extracellular-facing H⁺ transfer glutamate becomes directly exposed to aqueous solution during the transport cycle, but the intracellular glutamate E203, Glu_in, is buried within the protein. Two regions, denoted “polar” and “interfacial,” at the intracellular surface of the bacterial antiporter CLC-ec1 are examined here as possible pathways by which intracellular aqueous protons gain access to Glu_in. Mutations at multiple residues of the polar region have little effect on antiport rates. In contrast, mutation of E202, a conserved glutamate at the protein–water boundary of the interfacial region, leads to severe slowing of the Cl⁻/H⁺ antiport rate. An X-ray crystal structure of E202Y, the most strongly inhibited of these substitutions, shows an aqueous portal leading to Glu_in physically blocked by cross-subunit interactions; moreover, this mutation has only minimal effect on a monomeric CLC variant, which necessarily lacks such interactions. The several lines of experiments presented argue that E202 acts as a water-organizer that creates a proton conduit connecting intracellular solvent with Glu_in.     

The Late Endosomal ClC-6 Mediates Proton/Chloride Countertransport in Heterologous Plasma Membrane Expression

Members of the CLC protein family of Cl⁻ channels and transporters display the remarkable ability to function as either chloride channels or Cl⁻/H⁺ antiporters. Due to the intracellular localization of ClC-6 and ClC-7, it has not yet been possible to study the biophysical properties of these members of the late endosomal/lysosomal CLC branch in heterologous expression. Whereas recent data suggest that ClC-7 functions as an antiporter, transport characteristics of ClC-6 have remained entirely unknown. Here, we report that fusing the green fluorescent protein (GFP) to the N terminus of ClC-6 increased its cell surface expression, allowing us to functionally characterize ClC-6. Compatible with ClC-6 mediating Cl⁻/H⁺ exchange, Xenopus oocytes expressing GFP-tagged ClC-6 alkalinized upon depolarization. This alkalinization was dependent on the presence of extracellular anions and could occur against an electrochemical proton gradient. As observed in other CLC exchangers, ClC-6-mediated H⁺ transport was abolished by mutations in either the “gating” or “proton” glutamate. Overexpression of GFP-tagged ClC-6 in CHO cells elicited small, outwardly rectifying currents with a Cl⁻ > I⁻ conductance sequence. Mutating the gating glutamate of ClC-6 yielded an ohmic anion conductance that was increased by additionally mutating the “anion-coordinating” tyrosine. Additionally changing the chloride-coordinating serine 157 to proline increased the NO₃⁻ conductance of this mutant. Taken together, these data demonstrate for the first time that ClC-6 is a Cl⁻/H⁺ antiporter.     

Conversion of the 2 Cl−/1 H+ antiporter ClC‐5 in a NO3−/H+ antiporter by a single point mutation

Several members of the CLC family are secondary active anion/proton exchangers, and not passive chloride channels. Among the exchangers, the endosomal ClC-5 protein that is mutated in Dent''s disease shows an extreme outward rectification that precludes a precise determination of its transport stoichiometry from measurements of the reversal potential. We developed a novel imaging method to determine the absolute proton flux in Xenopus oocytes from the extracellular proton gradient. We determined a transport stoichiometry of 2 Cl⁻/1 H⁺. Nitrate uncoupled proton transport but mutating the highly conserved serine 168 to proline, as found in the plant NO₃⁻/H⁺ antiporter atClCa, led to coupled NO₃⁻/H⁺ exchange. Among several amino acids tested at position 168, S168P was unique in mediating highly coupled NO₃⁻/H⁺ exchange. We further found that ClC-5 is strongly stimulated by intracellular protons in an allosteric manner with an apparent pK of ∼7.2. A 2:1 stoichiometry appears to be a general property of CLC anion/proton exchangers. Serine 168 has an important function in determining anionic specificity of the exchange mechanism.     

Proton Transport Pathway in the ClC Cl/H Antiporter

A fundamental question concerning the ClC Cl⁻/H⁺ antiporters is the nature of their proton transport (PT) pathway. We addressed this issue by using a novel computational methodology capable of describing the explicit PT dynamics in the ClC-ec1 protein. The main result is that the Glu²⁰³ residue delivers a proton from the intracellular solution to the core of ClC-ec1 via a rotation of its side chain and subsequent acid dissociation. After reorientation of the Glu²⁰³ side chain, a transient water-mediated PT pathway between Glu²⁰³ and Glu¹⁴⁸ is established that is able to receive and translocate the proton via Grotthuss shuttling after deprotonation of Glu²⁰³. A molecular-dynamics simulation of an explicit hydrated excess proton in this pathway suggests that a negatively charged Glu¹⁴⁸ and the central Cl⁻ ion act together to drive H⁺ to the extracellular side of the membrane. This finding is consistent with the experimental result that Cl⁻ binding to the central site facilitates the proton movement. A calculation of the PT free-energy barrier for the ClC-ec1 E203V mutant also supports the proposal that a dissociable residue is required at this position for efficient delivery of H⁺ to the protein interior, in agreement with recent experimental results.     

CLC channels and transporters: Proteins with borderline personalities

Controlled chloride movement across membranes is essential for a variety of physiological processes ranging from salt homeostasis in the kidneys to acidification of cellular compartments. The CLC family is formed by two, not so distinct, sub-classes of membrane transport proteins: Cl^- channels and H⁺/Cl^- exchangers. 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 β-synthetase (CBS) domains. The C-termini are critical regulators of protein trafficking and directly modulate Cl- by binding intracellular ATP, H+ or oxidizing compounds. This review focuses on the recent mechanistic insights on the how the structural similarities between CLC channels and transporters translate in unexpected mechanistic analogies between these two sub-classes.     

Surprises from an Unusual CLC Homolog

The chloride channel (CLC) family is distinctive in that some members are Cl⁻ ion channels and others are Cl⁻/H⁺ antiporters. The molecular mechanism that couples H⁺ and Cl⁻ transport in the antiporters remains unknown. Our characterization of a novel bacterial homolog from Citrobacter koseri, CLC-ck2, has yielded surprising discoveries about the requirements for both Cl⁻ and H⁺ transport in CLC proteins. First, even though CLC-ck2 lacks conserved amino acids near the Cl⁻-binding sites that are part of the CLC selectivity signature sequence, this protein catalyzes Cl⁻ transport, albeit slowly. Ion selectivity in CLC-ck2 is similar to that in CLC-ec1, except that SO₄²⁻ strongly competes with Cl⁻ uptake through CLC-ck2 but has no effect on CLC-ec1. Second, and even more surprisingly, CLC-ck2 is a Cl⁻/H⁺ antiporter, even though it contains an isoleucine at the Glu_in position that was previously thought to be a critical part of the H⁺ pathway. CLC-ck2 is the first known antiporter that contains a nonpolar residue at this position. Introduction of a glutamate at the Glu_in site in CLC-ck2 does not increase H⁺ flux. Like other CLC antiporters, mutation of the external glutamate gate (Glu_ex) in CLC-ck2 prevents H⁺ flux. Hence, Glu_ex, but not Glu_in, is critical for H⁺ permeation in CLC proteins.The chloride channel (CLC) family includes both Cl⁻ ion channels and Cl⁻/H⁺ antiporters (1). The ion channels allow Cl⁻ to diffuse passively down an electrochemical gradient, and antiporters couple the movement of chloride and protons in opposite directions across cellular membranes. So far, the only known CLC structures are those of antiporters (2–4). On the basis of sequence similarity and functional studies, it is thought that the basic structures of the ion channels and antiporters are similar, and that slight structural differences account for these diverse functions. Understanding how the CLC family has evolved to allow proteins of similar structure to carry out two distinct mechanisms remains a critical goal.In the Escherichia coli antiporter CLC-ec1, two glutamates, Glu_ex (E148) and Glu_in (E203), are absolutely required for H⁺ transport (5,6). Glu_ex is conserved in both CLC ion channels and antiporters. Glu_in is conserved only in antiporters and is instead a hydrophobic valine in all of the known ion channels. Hence, it was proposed that both Glu_in and Glu_ex are necessary to transfer protons through CLC antiporters (6). Studies of the CLC-4 and CLC-5 antiporters supported the notion that Glu_in and Glu_ex play critical roles in H⁺ transport (7,8). Surprisingly, however, recent experiments revealed that although the red algae homolog CmCLC contains a threonine at the Glu_in position, it is still Cl⁻/H⁺ antiporter (3). It is unknown whether this threonine has a shifted pKa that allows it to transfer protons or whether the H⁺ transport in CmCLC does not require a protonatable residue at this position. Further blurring the role of Glu_in, the CLC-0 ion channel, which contains a valine at the Glu_in position, requires slow transmembrane H⁺ transport for channel gating (9).To probe the molecular requirements for Cl⁻ and H⁺ transport in CLC proteins, we characterized a novel homolog from Citrobacter koseri called CLC-ck2. CLC-ck2 is 21% identical and 37% similar in amino acid sequence to CLC-ec1. CLC-ck2 contains an isoleucine at the Glu_in position, and hence we originally hypothesized that this protein would act as an ion channel. Additionally, CLC-ck2 lacks several amino acids that coordinate the central and internal Cl⁻-binding sites in CLC-ec1, most notably the GSGIP motif (Fig. S1 in the Supporting Material). With genomic information now revealing >1000 putative CLC homologs, we find that CLC-ck2 is not unique—several other uncharacterized homologs also lack these regions. To our knowledge, ours is the first study to characterize the function of a homolog missing these regions.Using Cl⁻ flux assays, we first sought to determine whether CLC-ck2 could catalyze Cl⁻ transport (10). With CLC-ck2-containing vesicles, slow but significant Cl⁻ efflux was observed upon addition of valinomycin (Vln; Fig. 1 A, blue trace). In control vesicles lacking CLC-ck2, no significant Cl⁻ flux was observed (Fig. 1 A, black). The CLC-ec1 inhibitor 4,4′-octanamidostilbene-2,2′-disulfonate (OADS) (11) completely inhibited Cl⁻ flux (Fig. 1 A, green). The Cl⁻ unitary turnover rate for wild-type CLC-ck2 was 31 ± 5 s⁻¹ (mean ± SE, n = 5). This rate is ∼2 orders of magnitude less than the Cl⁻ flux through the CLC-ec1 antiporter, and is much slower transport than expected for an ion channel. However, it is a similar to the rate catalyzed by the cyanobacterium antiporter CLC-sy1 (4).Open in a separate windowFigure 1(A) Representative Cl⁻ flux assays. Cl⁻ efflux was initiated by addition of Vln. Triton X-100 was added to disrupt liposomes and release all intracellular Cl⁻. The insert shows an expanded view of the efflux immediately after addition of Vln. (B) Representative H⁺ flux assays demonstrate Cl⁻-driven H⁺ influx. H⁺ flux was initiated by the addition of Vln. The H⁺ gradient was collapsed at the end by the addition of FCCP.To test whether CLC-ck2 is a Cl⁻ ion channel or Cl⁻/H⁺ antiporter, we performed H⁺ flux assays as previously described (10). If vesicles contain a Cl⁻/H⁺ antiporter, the efflux of Cl⁻ upon addition of Vln will drive the movement of protons into the vesicles against their concentration gradient. If vesicles contain a Cl⁻ ion channel, however, no movement of protons will be observed upon addition of Vln. We found that CLC-ck2 showed significant Cl⁻-driven H⁺ uptake. Fig. 1 B illustrates uphill movement of protons in the presence of a Cl⁻ gradient. H⁺ influx, like Cl⁻ efflux, was inhibited by the presence of OADS. These assays are not quantitative enough to determine Cl⁻/H⁺ stoichiometry. However, they qualitatively demonstrate that CLC-ck2 acts as a Cl⁻/H⁺ antiporter even though it lacks Glu_in.If Glu_in is important for maximizing H⁺ flux, we would expect that introducing a glutamate at the Glu_in position would increase the H⁺ flux observed through CLC-ck2. However, we found that the I175E mutation did not significantly alter H⁺ or Cl⁻ flux (Fig. 2). Hence, Glu_in does not enhance H⁺ transport through CLC-ck2.Open in a separate windowFigure 2Unitary turnover rates, calculated from initial velocities after addition of Vln in (A) Cl⁻ and (B) H⁺ flux assays. Reconstitutions contained 5–38 μg protein/mg lipid. Bars represent the mean ± SE for three to 17 assays.The external glutamate gate Glu_ex is conserved and required for H⁺ transport in all known CLC antiporters (5,7). To determine whether Glu_ex is also essential for H⁺ transport in CLC-ck2, we made the E122Q mutation. This mutant can still transport chloride but fails to move protons (Fig. 2). This mutant protein was not very stable in micelles, precipitating over the course of hours, and thus the unitary turnover rates shown in Fig. 2 represent lower limits. Nevertheless, this result is consistent with observations in other CLC antiporters and suggests that Glu_ex is important for H⁺ transport in all CLC antiporters.Because CLC-ck2 lacks amino acids that coordinate the Cl⁻ ions in the structure of CLC-ec1, we wondered whether the ion selectivity might differ. Indeed, the plant atCLC-a homolog has a single change in this region that makes it selective for NO₃⁻ over Cl⁻ (12). To determine the ion selectivity of CLC-ck2, we used radioactive uptake assays (11). In these assays, the amount of ³⁶Cl⁻ exchanged into CLC-ck2-containing vesicles loaded with cold Cl⁻ is measured as a function of time. Various anions were added to the extravesicular solution to test which ions were transported in preference to the ³⁶Cl⁻. A decrease in radioactive uptake indicates that the anion is permeant and/or blocks CLC-ck2. Fig. 3 A plots the amount of ³⁶Cl⁻ uptake with each of the various ions added; the ion selectivity (or block) was SO₄²⁻ ≫ Cl⁻ > NO₃⁻ > SCN⁻ >Br⁻ > F⁻ > P_i⁻ ≈ I⁻ ≫ isethionate⁻. This selectivity is similar to that of CLC-ec1 (13), with one noticeable exception: SO₄²⁻. SO₄²⁻ had no effect on CLC-ec1, but strongly competed with Cl⁻ uptake through CLC-ck2 (Fig. 3 B). Hence, the selectivity filter of CLC-ck2 is similar enough to other CLCs to transport Cl⁻, NO₃⁻, and Br⁻ as expected. However, further investigation is required to determine the structural differences that must underlie the distinct disparity in SO₄²⁻ permeability and/or block.Open in a separate windowFigure 3Ion selectivity of CLC-ck2. (A) Liposomes reconstituted with CLC-ck2 were screened for selectivity against various test ions in the presence of 1 mM ³⁶Cl⁻ at pH 4.5. All test ions were present at 10 mM, except for isethionate, which was present at 20 mM to confirm that it is inert. After 10 min, the radioactivity counts were measured to determine total ³⁶Cl⁻ uptake (for isethionate, uptake was stopped after 20 min). Counts were normalized with respect to liposome uptake in the absence of an external test ion. Bars represent the mean ± SE for three assays. (B) Comparison of effects of external sulfate on CLC-ec1 and CLC-ck2 on radioactive update assays, normalized as in part A.This study reveals that Glu_in is not essential for Cl⁻- coupled H⁺ transport in CLC-ck2, in direct contrast to the previous conclusion that the protonatable side chain of the glutamate is directly involved in the H⁺ transport pathway (14). Thus, our result brings into question the location of the H⁺ permeation pathway. The protons must be transferred via other protonatable residues or water molecules. The residue adjacent to Glu_in (E202 in CLC-ec1) is conserved in CLC-ck2. Unfortunately, mutation of this glutamate (E174F) in CLC-ck2 resulted in unstable protein that could not be characterized in functional studies. Using the structure of CLC-ec1 as a guide, we see no other obvious protonatable residues in CLC-ck2 available to transfer protons from the intracellular side to Glu_ex. One possibility is that H⁺ transport may require a water wire. The idea of a water wire is not new. In CLC-ec1, there is an ∼15 Å gap between Glu_in and Glu_ex, and it has never been clear exactly how protons cross this gap. Recent molecular-dynamics studies have supported the idea that the Glu_in in CLC-ec1 may help to position water molecules for a water wire to transfer protons to the extracellular glutamate (15). If indeed the role of Glu_in is simply to position water molecules properly to transfer protons, subtle changes in other parts of the structure could allow this water wire to exist in the absence of Glu_in. This could also explain how the eukaryotic CmCLC homolog, which has a threonine at the Glu_in position, is able to act as a coupled transporter as well. We have not yet been able to determine the structure of CLC-ck2 to understand how the lack of conserved amino acids near the Cl⁻-binding sites affects the structure. This study will inspire future work to investigate the molecular mechanism of CLC-ck2 and CLC-ck2 homologs in greater detail.     

Intracellular regulation of human ClC‐5 by adenine nucleotides

ClC-5, an endosomal Cl⁻/H⁺ antiporter that is mutated in Dent disease, is essential for endosomal acidification and re-uptake of small molecular weight proteins in the renal proximal tubule. Eukaryotic chloride channels (CLCs) contain two cytoplasmic CBS domains, motifs present in different proteins, the function of which is still poorly understood. Structural studies have shown that ClC-5 can bind to ATP at the interface between the CBS domains, but so far the potential functional consequences of nucleotide binding to ClC-5 have not been investigated. Here, we show that the direct application of ATP, ADP and AMP in inside-out patch experiments potentiates the current mediated by ClC-5 with similar affinities. The nucleotides increase the probability of ClC-5 to be in an active, transporting state. The residues Tyr 617 and Asp 727, but not Ser 618, are crucial for the potentiation. These results provide a mechanistic and structural framework for the interpretation of nucleotide regulation of a CLC transporter.     

Residues Important for Nitrate/Proton Coupling in Plant and Mammalian CLC Transporters

Members of the CLC gene family either function as chloride channels or as anion/proton exchangers. The plant AtClC-a uses the pH gradient across the vacuolar membrane to accumulate the nutrient in this organelle. When AtClC-a was expressed in Xenopus oocytes, it mediated exchange and less efficiently mediated Cl^–/H⁺ exchange. Mutating the “gating glutamate” Glu-203 to alanine resulted in an uncoupled anion conductance that was larger for Cl^– than . Replacing the “proton glutamate” Glu-270 by alanine abolished currents. These could be restored by the uncoupling E203A mutation. Whereas mammalian endosomal ClC-4 and ClC-5 mediate stoichiometrically coupled 2Cl^–/H⁺ exchange, their transport is largely uncoupled from protons. By contrast, the AtClC-a-mediated accumulation in plant vacuoles requires tight coupling. Comparison of AtClC-a and ClC-5 sequences identified a proline in AtClC-a that is replaced by serine in all mammalian CLC isoforms. When this proline was mutated to serine (P160S), Cl^–/H⁺ exchange of AtClC-a proceeded as efficiently as exchange, suggesting a role of this residue in exchange. Indeed, when the corresponding serine of ClC-5 was replaced by proline, this Cl^–/H⁺ exchanger gained efficient coupling. When inserted into the model Torpedo chloride channel ClC-0, the equivalent mutation increased nitrate relative to chloride conductance. Hence, proline in the CLC pore signature sequence is important for exchange and conductance both in plants and mammals. Gating and proton glutamates play similar roles in bacterial, plant, and mammalian CLC anion/proton exchangers.CLC proteins are found in all phyla from bacteria to humans and either mediate electrogenic anion/proton exchange or function as chloride channels (1). In mammals, the roles of plasma membrane CLC Cl^– channels include transepithelial transport (2–5) and control of muscle excitability (6), whereas vesicular CLC exchangers may facilitate endocytosis (7) and lysosomal function (8–10) by electrically shunting vesicular proton pump currents (11). In the plant Arabidopsis thaliana, there are seven CLC isoforms (AtClC-a–AtClC-g)² (12–15), which may mostly reside in intracellular membranes. AtClC-a uses the pH gradient across the vacuolar membrane to transport the nutrient nitrate into that organelle (16). This secondary active transport requires a tightly coupled exchange. Astonishingly, however, mammalian ClC-4 and -5 and bacterial EcClC-1 (one of the two CLC isoforms in Escherichia coli) display tightly coupled Cl^–/H⁺ exchange, but anion flux is largely uncoupled from H⁺ when is transported (17–21). The lack of appropriate expression systems for plant CLC transporters (12) has so far impeded structure-function analysis that may shed light on the ability of AtClC-a to perform efficient exchange. This dearth of data contrasts with the extensive mutagenesis work performed with CLC proteins from animals and bacteria.The crystal structure of bacterial CLC homologues (22, 23) and the investigation of mutants (17, 19–21, 24–29) have yielded important insights into their structure and function. CLC proteins form dimers with two largely independent permeation pathways (22, 25, 30, 31). Each of the monomers displays two anion binding sites (22). A third binding site is observed when a certain key glutamate residue, which is located halfway in the permeation pathway of almost all CLC proteins, is mutated to alanine (23). Mutating this gating glutamate in CLC Cl^– channels strongly affects or even completely suppresses single pore gating (23), whereas CLC exchangers are transformed by such mutations into pure anion conductances that are not coupled to proton transport (17, 19, 20). Another key glutamate, located at the cytoplasmic surface of the CLC monomer, seems to be a hallmark of CLC anion/proton exchangers. Mutating this proton glutamate to nontitratable amino acids uncouples anion transport from protons in the bacterial EcClC-1 protein (27) but seems to abolish transport altogether in mammalian ClC-4 and -5 (21). In those latter proteins, anion transport could be restored by additionally introducing an uncoupling mutation at the gating glutamate (21).The functional complementation by AtClC-c and -d (12, 32) of growth phenotypes of a yeast strain deleted for the single yeast CLC Gef1 (33) suggested that these plant CLC proteins function in anion transport but could not reveal details of their biophysical properties. We report here the first functional expression of a plant CLC in animal cells. Expression of wild-type (WT) and mutant AtClC-a in Xenopus oocytes indicate a general role of gating and proton glutamate residues in anion/proton coupling across different isoforms and species. We identified a proline in the CLC signature sequence of AtClC-a that plays a crucial role in exchange. Mutating it to serine, the residue present in mammalian CLC proteins at this position, rendered AtClC-a Cl^–/H⁺ exchange as efficient as exchange. Conversely, changing the corresponding serine of ClC-5 to proline converted it into an efficient exchanger. When proline replaced the critical serine in Torpedo ClC-0, the relative conductance of this model Cl^– channel was drastically increased, and “fast” protopore gating was slowed.     

The CLC 'chloride channel' family: revelations from prokaryotes

Members of the CLC 'chloride channel' family play vital roles in a wide variety of physiological settings. Research on prokaryotic CLC homologues provided long-anticipated high-resolution structures as well as the unexpected discovery that some CLCs are not chloride channels, but rather are proton-chloride antiporters. Hence, CLCs encompass two functional classes of transport proteins once thought to be fundamentally different from one another. In this review, we discuss the structural features and molecular mechanisms of CLC channels and antiporters. We focus on ClC-0, the most thoroughly studied CLC channel, and ClC-ec1, the prokaryotic antiporter of known structure. We highlight some striking similarities between these CLCs and discuss compelling questions that remain to be addressed. Prokaryotic CLCs will undoubtedly continue to shed light upon this understudied family of proteins.     

The CLC ‘chloride channel’ family: revelations from prokaryotes (Review)

Members of the CLC ‘chloride channel’ family play vital roles in a wide variety of physiological settings. Research on prokaryotic CLC homologues provided long-anticipated high-resolution structures as well as the unexpected discovery that some CLCs are not chloride channels, but rather are proton-chloride antiporters. Hence, CLCs encompass two functional classes of transport proteins once thought to be fundamentally different from one another. In this review, we discuss the structural features and molecular mechanisms of CLC channels and antiporters. We focus on ClC-0, the most thoroughly studied CLC channel, and ClC-ec1, the prokaryotic antiporter of known structure. We highlight some striking similarities between these CLCs and discuss compelling questions that remain to be addressed. Prokaryotic CLCs will undoubtedly continue to shed light upon this understudied family of proteins.     

Extracellular Chloride Regulates the Epithelial Sodium Channel

The extracellular domain of the epithelial sodium channel ENaC is exposed to a wide range of Cl⁻ concentrations in the kidney and in other epithelia. We tested whether Cl⁻ alters ENaC activity. In Xenopus oocytes expressing human ENaC, replacement of Cl⁻ with SO₄²⁻, H₂PO₄⁻, or SCN⁻ produced a large increase in ENaC current, indicating that extracellular Cl⁻ inhibits ENaC. Extracellular Cl⁻ also inhibited ENaC in Na⁺-transporting epithelia. The anion selectivity sequence was SCN⁻ < SO₄²⁻ < H₂PO₄⁻ < F⁻ < I⁻ < Cl⁻ < Br⁻. Crystallization of ASIC1a revealed a Cl⁻ binding site in the extracellular domain. We found that mutation of corresponding residues in ENaC (α_H418A and β_R388A) disrupted the response to Cl⁻, suggesting that Cl⁻ might regulate ENaC through an analogous binding site. Maneuvers that lock ENaC in an open state (a DEG mutation and trypsin) abolished ENaC regulation by Cl⁻. The response to Cl⁻ was also modulated by changes in extracellular pH; acidic pH increased and alkaline pH reduced ENaC inhibition by Cl⁻. Cl⁻ regulated ENaC activity in part through enhanced Na⁺ self-inhibition, a process by which extracellular Na⁺ inhibits ENaC. Together, the data indicate that extracellular Cl⁻ regulates ENaC activity, providing a potential mechanism by which changes in extracellular Cl⁻ might modulate epithelial Na⁺ absorption.The epithelial Na⁺ channel ENaC² is a heterotrimer of homologous α, β, and γ subunits (1, 2). ENaC functions as a pathway for Na⁺ absorption across epithelial cells in the kidney collecting duct, lung, distal colon, and sweat duct (reviewed in Refs. 3 and 4). Na⁺ transport is critical for the maintenance of Na⁺ homeostasis and for the control of the composition and quantity of the fluid on the apical membrane of these epithelia. ENaC mutations and defects in its regulation cause inherited forms of hypertension and hypotension (5) and may contribute to the pathogenesis of lung disease in cystic fibrosis (6).ENaC is a member of the DEG/ENaC family of ion channels. A common structural feature of these channels is a large extracellular domain that plays a critical role in channel gating. For example, in ASICs, the extracellular domain functions as a receptor for protons, which transiently activate the channel by titrating residues that form an acidic pocket (7). FaNaCh is a ligand-gated family member in Helix aspersa, activated by the peptide FMRFamide (8). In Caenorhabditis elegans MEC family members, the extracellular domain is thought to respond to mechanical signals (9).ENaC differs from other family members because it is constitutively active in the absence of a ligand/stimulus. However, a convergence of data indicate that ENaC gating is modulated by a variety of molecules that bind to or modify its extracellular domains, including proteases (10–12), Na⁺ (13–15), protons (16), and the divalent cations Zn²⁺ and Ni²⁺ (17, 18). These findings suggest that the ENaC extracellular domain might regulate epithelial Na⁺ transport by sensing and integrating diverse signals in the extracellular environment.In the current study, we tested the hypothesis that ENaC activity is regulated by changes in the extracellular Cl⁻ concentration. Several observations suggested that Cl⁻ might be a strong candidate to regulate the channel. First, transport of Na⁺ and Cl⁻ are often coupled to maintain electroneutrality. Second, ENaC is exposed to large changes in extracellular Cl⁻ concentration. For example, in the kidney collecting duct, the urine Cl⁻ concentration varies widely (19). As the predominant anion, its concentration parallels that of Na⁺ in most clinical states. However, under conditions of metabolic alkalosis and metabolic acidosis, the Na⁺ and Cl⁻ concentrations can become dissociated as a result of increased urinary bicarbonate (alkalosis) or ammonium (acidosis) (19). Thus, ENaC is well positioned to respond to changes in Cl⁻ concentration. Third, crystallization of ASIC1a revealed a binding site for a Cl⁻ ion at the base of the thumb domain (7). The Cl⁻ is coordinated by Arg-310 and Glu-314 from one subunit and Lys-212 from an adjacent subunit. Although the functional role of Cl⁻ binding to ASIC1a is unknown, it supports the hypothesis that extracellular Cl⁻ might regulate the activity of DEG/ENaC ion channels.     

Interactions between permeation and gating in the TMEM16B/anoctamin2 calcium-activated chloride channel

At least two members of the TMEM16/anoctamin family, TMEM16A (also known as anoctamin1) and TMEM16B (also known as anoctamin2), encode Ca²⁺-activated Cl⁻ channels (CaCCs), which are found in various cell types and mediate numerous physiological functions. Here, we used whole-cell and excised inside-out patch-clamp to investigate the relationship between anion permeation and gating, two processes typically viewed as independent, in TMEM16B expressed in HEK 293T cells. The permeability ratio sequence determined by substituting Cl⁻ with other anions (P_X/P_Cl) was SCN⁻ > I⁻ > NO₃⁻ > Br⁻ > Cl⁻ > F⁻ > gluconate. When external Cl⁻ was substituted with other anions, TMEM16B activation and deactivation kinetics at 0.5 µM Ca²⁺ were modified according to the sequence of permeability ratios, with anions more permeant than Cl⁻ slowing both activation and deactivation and anions less permeant than Cl⁻ accelerating them. Moreover, replacement of external Cl⁻ with gluconate, or sucrose, shifted the voltage dependence of steady-state activation (G-V relation) to more positive potentials, whereas substitution of extracellular or intracellular Cl⁻ with SCN⁻ shifted G-V to more negative potentials. Dose–response relationships for Ca²⁺ in the presence of different extracellular anions indicated that the apparent affinity for Ca²⁺ at +100 mV increased with increasing permeability ratio. The apparent affinity for Ca²⁺ in the presence of intracellular SCN⁻ also increased compared with that in Cl⁻. Our results provide the first evidence that TMEM16B gating is modulated by permeant anions and provide the basis for future studies aimed at identifying the molecular determinants of TMEM16B ion selectivity and gating.     

The role of protons in fast and slow gating of the Torpedo chloride channel ClC-0

Transmembrane proton transport is of fundamental importance for life. The list of H⁺ transporting proteins has been recently expanded with the discovery that some members of the CLC gene family are stoichiometrically coupled Cl⁻/H⁺ antiporters. Other CLC proteins are instead passive Cl⁻ selective anion channels. The gating of these CLC channels is, however, strongly regulated by pH, likely reflecting the evolutionary relationship with CLC Cl⁻/H⁺ antiporters. The role of protons in the gating of the model Torpedo channel ClC-0 is best understood. 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. Extracellular protons likely can arrive directly at E166. In contrast, protonation of E166 from the inside has been proposed to be mediated by the dissociation of an intrapore water molecule. The OH⁻ anion resulting from the water dissociation is stabilized in one of the anion binding sites of the channel, competing with intracellular Cl⁻ ions. The pH dependence of the slow gate is less well understood. It has been shown that proton translocation drives irreversible gating transitions associated with the slow gate. However, the relationship of the fast gate’s pH dependence on the proton translocation and the molecular basis of the slow gate remain to be discovered.     

Multiscale Simulations Reveal Key Aspects of the Proton Transport Mechanism in the ClC-ec1 Antiporter

Multiscale reactive molecular dynamics simulations are used to study proton transport through the central region of ClC-ec1, a widely studied ClC transporter that enables the stoichiometric exchange of 2 Cl^– ions for 1 proton (H⁺). It has long been known that both Cl^– and proton transport occur through partially congruent pathways, and that their exchange is strictly coupled. However, the nature of this coupling and the mechanism of antiporting remain topics of debate. Here multiscale simulations have been used to characterize proton transport between E203 (Glu_in) and E148 (Glu_ex), the internal and external intermediate proton binding sites, respectively. Free energy profiles are presented, explicitly accounting for the binding of Cl^– along the central pathway, the dynamically coupled hydration changes of the central region, and conformational changes of Glu_in and Glu_ex. We find that proton transport between Glu_in and Glu_ex is possible in both the presence and absence of Cl^– in the central binding site, although it is facilitated by the anion presence. These results support the notion that the requisite coupling between Cl^– and proton transport occurs elsewhere (e.g., during proton uptake or release). In addition, proton transport is explored in the E203K mutant, which maintains proton permeation despite the substitution of a basic residue for Glu_in. This collection of calculations provides for the first time, to our knowledge, a detailed picture of the proton transport mechanism in the central region of ClC-ec1 at a molecular level.     

CLC transport proteins in plants

Nitrate compartmentalization in intracellular organelles has been long recognized as critical for plant physiology but the molecular identity of the proteins involved remained unclear for a long time. In Arabidopsis thaliana, AtClC-a has been recently shown to be a antiporter critical for nitrate transport into the vacuoles. AtClC-a is a member of the CLC protein family, whose animal and bacterial members, comprising both channels and H⁺-coupled antiporters, have been previously implicated exclusively in Cl⁻ transport. Despite the different over Cl⁻ selectivity of AtClC-a compared to the other CLC antiporters, it has similar transport properties.Other CLC homologues have been cloned in Arabidopsis, tobacco, rice and soybean.     

Mechanism of Ion Permeation in Skeletal Muscle Chloride Channels

Voltage-gated Cl⁻ channels belonging to the ClC family exhibit unique properties of ion permeation and gating. We functionally probed the conduction pathway of a recombinant human skeletal muscle Cl⁻ channel (hClC-1) expressed both in Xenopus oocytes and in a mammalian cell line by investigating block by extracellular or intracellular I⁻ and related anions. Extracellular and intracellular I⁻ exert blocking actions on hClC-1 currents that are both concentration and voltage dependent. Similar actions were observed for a variety of other halide (Br⁻) and polyatomic (SCN⁻, NO₃ ⁻, CH₃SO₃ ⁻) anions. In addition, I⁻ block is accompanied by gating alterations that differ depending on which side of the membrane the blocker is applied. External I⁻ causes a shift in the voltage-dependent probability that channels exist in three definable kinetic states (fast deactivating, slow deactivating, nondeactivating), while internal I⁻ slows deactivation. These different effects on gating properties can be used to distinguish two functional ion binding sites within the hClC-1 pore. We determined K _D values for I⁻ block in three distinct kinetic states and found that binding of I⁻ to hClC-1 is modulated by the gating state of the channel. Furthermore, estimates of electrical distance for I⁻ binding suggest that conformational changes affecting the two ion binding sites occur during gating transitions. These results have implications for understanding mechanisms of ion selectivity in hClC-1, and for defining the intimate relationship between gating and permeation in ClC channels.     

Multiple Discrete Transitions Underlie Voltage-Dependent Activation in CLC Cl/H Antiporters

Most mammalian chloride channels and transporters in the CLC family display pronounced voltage-dependent gating. Surprisingly, despite the complex nature of the gating process and the large contribution to it by the transport substrates, experimental investigations of the fast gating process usually produce canonical Boltzmann activation curves that correspond to a simple two-state activation. By using nonlinear capacitance measurements of two mutations in the ClC-5 transporter, here we are able to discriminate and visualize discrete transitions along the voltage-dependent activation pathway. The strong and specific dependence of these transitions on internal and external [Cl⁻] suggest that CLC gating involves voltage-dependent conformational changes as well as coordinated movement of transported substrates.     

Functional Architecture of the Cytoplasmic Entrance to the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore

As an ion channel, the cystic fibrosis transmembrane conductance regulator must form a continuous pathway for the movement of Cl⁻ and other anions between the cytoplasm and the extracellular solution. Both the structure and the function of the membrane-spanning part of this pathway are well defined. In contrast, the structure of the pathway that connects the cytoplasm to the membrane-spanning regions is unknown, and functional roles for different parts of the protein forming this pathway have not been described. We used patch clamp recording and substituted cysteine accessibility mutagenesis to identify positively charged amino acid side chains that attract cytoplasmic Cl⁻ ions to the inner mouth of the pore. Our results indicate that the side chains of Lys-190, Arg-248, Arg-303, Lys-370, Lys-1041, and Arg-1048, located in different intracellular loops of the protein, play important roles in the electrostatic attraction of Cl⁻ ions. Mutation and covalent modification of these residues have charge-dependent effects on the rate of Cl⁻ permeation, demonstrating their functional role in maximization of Cl⁻ flux. Other nearby positively charged side chains were not involved in electrostatic interactions with Cl⁻. The location of these Cl⁻-attractive residues suggests that cytoplasmic Cl⁻ ions enter the pore via a lateral portal located between the cytoplasmic extensions to the fourth and sixth transmembrane helices; a secondary, functionally less relevant portal might exist between the extensions to the 10th and 12th transmembrane helices. These results define the cytoplasmic mouth of the pore and show how it attracts Cl⁻ ions from the cytoplasm.     

A Novel Role of Protein Tyrosine Kinase2 in Mediating Chloride Secretion in Human Airway Epithelial Cells

Ca²⁺ activated Cl⁻ channels (CaCC) are up-regulated in cystic fibrosis (CF) airway surface epithelia. The presence and functional properties of CaCC make it a possible therapeutic target to compensate for the deficiency of Cl⁻ secretion in CF epithelia. CaCC is activated by an increase in cytosolic Ca²⁺, which not only activates epithelial CaCCs, but also inhibits epithelial Na⁺ hyperabsorption, which may also be beneficial in CF. Our previous study has shown that spiperone, a known antipsychotic drug, activates CaCCs and stimulates Cl⁻ secretion in polarized human non-CF and CF airway epithelial cell monolayers in vitro, and in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) knockout mice in vivo. Spiperone activates CaCC not by acting in its well-known role as an antagonist of either 5-HT2 or D2 receptors, but through a protein tyrosine kinase-coupled phospholipase C-dependent pathway. Moreover, spiperone independently activates CFTR through a novel mechanism. Herein, we performed a mass spectrometry analysis and identified the signaling molecule that mediates the spiperone effect in activating chloride secretion through CaCC and CFTR. Proline-rich tyrosine kinase 2 (PYK2) is a non-receptor protein tyrosine kinase, which belongs to the focal adhesion kinase family. The inhibition of PYK2 notably reduced the ability of spiperone to increase intracellular Ca²⁺ and Cl⁻ secretion. In conclusion, we have identified the tyrosine kinase, PYK2, as the modulator, which plays a crucial role in the activation of CaCC and CFTR by spiperone. The identification of this novel role of PYK2 reveals a new signaling pathway in human airway epithelial cells.