Exterior site occupancy infers chloride-induced proton gating in a prokaryotic homolog of the ClC chloride channel

The ClC family of anion channels mediates the efficient, selective permeation of Cl(-) across the biological membranes of living cells under the driving force of an electrochemical gradient. In some eukaryotes, these channels are known to exhibit a unique gating mechanism, which appears to be triggered by the permeant Cl(-) anion. We infer details of this gating mechanism by studying the free energetics of Cl(-) occupancy in the pore of a prokaryotic ClC homolog. These free energetics were gleaned from 30 ns of molecular dynamics simulation on an approximately 133,000-atom system consisting of a hydrated membrane embedded StClC transporter. The binding sites for Cl(-) in the transporter were determined for the cases where the putative gating residue, Glu(148), was protonated and unprotonated. When the glutamate gate is protonated, Cl(-) favorably occupies an exterior site, S(ext), to form a queue of anions in the pore. However, when the glutamate gate is unprotonated, Cl(-) cannot occupy this site nor, consequently, pass through the pore. An additional, previously undetected, site was found in the pore near the outer membrane that exists regardless of the protonation state of Glu(148). Although this suggests that, for the prokaryotic homolog, protonation of Glu(148) may be the first step in transporting Cl(-) at the expense of H(+) transport in the opposite direction, an evolutionary argument might suggest that Cl(-) opens the ClC gate in eukaryotic channels by inducing the conserved glutamate's protonation. During an additional 20 ns free dynamics simulation, the newly discovered outermost site, S(out), and the innermost site, S(int), were seen to allow spontaneous exchange of Cl(-) ions with the bulk electrolyte while under depolarization conditions.     

Structural basis for ion conduction and gating in ClC chloride channels

Members of the ClC family of voltage-gated chloride channels are found from bacteria to mammals with a considerable degree of conservation in the membrane-inserted, pore-forming region. The crystal structures of the ClC channels of Escherichia coli and Salmonella typhimurium provide a structural framework for the entire family. The ClC channels are homodimeric proteins with an overall rhombus-like shape. Each ClC dimer has two pores each contained within a single subunit. The ClC subunit consists of two roughly repeated halves that span the membrane with opposite orientations. This antiparallel architecture defines a chloride selectivity filter within the 15-A neck of a hourglass-shaped pore. Three Cl(-) binding sites within the selectivity filter stabilize ions by interactions with alpha-helix dipoles and by chemical interactions with nitrogen atoms and hydroxyl groups of residues in the protein. The Cl(-) binding site nearest the extracellular solution can be occupied either by a Cl(-) ion or by a glutamate carboxyl group. Mutations of this glutamate residue in Torpedo ray ClC channels alter gating in electrophysiological assays. These findings reveal a form of gating in which the glutamate carboxyl group closes the pore by mimicking a Cl(-) ion.     

Roles of K149, G352, and H401 in the channel functions of ClC-0: testing the predictions from theoretical calculations

The ClC family of Cl(-) channels and transporters comprises membrane proteins ubiquitously present in species ranging from prokaryotes to mammals. The recently solved structures of the bacterial ClC proteins have provided a good model to guide the functional experiments for the eukaryotic Cl(-) channels. Theoretical calculations based on the bacterial ClC structures have identified several residues critical for the Cl(-) binding energy in the Cl(-) transport pathway. It was speculated that the corresponding residues in eukaryotic Cl(-) channels might play similar roles for the channel functions. In this study, we made a series of mutations in three such residues in eukaryotic ClC Cl(-) channels (K149, G352, and H401 in ClC-0) and studied the functional consequences on the channel properties. A cysteine modification approach was also employed to evaluate the electrostatic effects of the charge placed at these three positions. The experimental results revealed that among the three residues tested, K149 plays the most important role in controlling both the gating and the permeation functions of ClC-0. On the other hand, mutations of H401 alter the channel conductance but not the gating properties, while mutations of G352 result in very little functional consequence. The mutation of K149 into a neutral residue leucine (K149L) shifts the activation curve and leads to flickery channel openings. The anion permeability ratios derived from bi-ionic experiments are also significantly altered in that the selectivity of Cl(-) over other anions is decreased. Furthermore, removing the positive charge at this position reduces and increases, respectively, the accessibility of the negatively and positively charged methane thiosulfonate reagents to the pore. The control of the accessibility to charged MTS reagents and the regulation of the anion permeation support the idea that K149 exerts an electrostatic effect on the channel function, confirming the prediction from computational studies.     

A synthetic chloride channel restores chloride conductance in human cystic fibrosis epithelial cells

Mutations in the gene-encoding cystic fibrosis transmembrane conductance regulator (CFTR) cause defective transepithelial transport of chloride (Cl(-)) ions and fluid, thereby becoming responsible for the onset of cystic fibrosis (CF). One strategy to reduce the pathophysiology associated with CF is to increase Cl(-) transport through alternative pathways. In this paper, we demonstrate that a small synthetic molecule which forms Cl(-) channels to mediate Cl(-) transport across lipid bilayer membranes is capable of restoring Cl(-) permeability in human CF epithelial cells; as a result, it has the potential to become a lead compound for the treatment of human diseases associated with Cl(-) channel dysfunction.     

Glutamate 268 regulates transport probability of the anion/proton exchanger ClC-5

The Cl(-)/H(+) exchange mediated by ClC transporters can be uncoupled by external SCN(-) and mutations of the proton glutamate, a conserved residue at the internal side of the protein. We show here for the mammalian ClC transporter ClC-5 that acidic internal pH led to a greater increase in currents upon exchanging extracellular Cl(-) for SCN(-). However, transport uncoupling, unitary current amplitudes, and the voltage dependence of the depolarization-induced activation were not altered by low pH values. Therefore, it is likely that an additional gating process regulates ClC-5 transport. Higher internal [H(+)] and the proton glutamate mutant E268H altered the ratio between ClC-5 transport and nonlinear capacitance, indicating that the gating charge movements in ClC-5 arise from incomplete transport cycles and that internal protons increase the transport probability of ClC-5. This was substantiated by site-directed sulfhydryl modification of the proton glutamate mutant E268C. The mutation exhibited small transport currents together with prominent gating charge movements. The charge restoration using a negatively charged sulfhydryl reagent reinstated also the WT phenotype. Neutralization of the charge of the gating glutamate 211 by the E211C mutation abolished the effect of internal protons, showing that the increased transport probability of ClC-5 results from protonation of this residue. S168P (a mutation that decreases the anion affinity of the central binding site) reduced also the internal pH dependence of ClC-5. These results support the idea that protonation of the gating glutamate 211 at the central anion-binding site of ClC-5 is mediated by the proton glutamate 268.     

Unique gating properties of C. elegans ClC anion channel splice variants are determined by altered CBS domain conformation and the R-helix linker

All eukaryotic and some prokaryotic ClC anion transport proteins have extensive cytoplasmic C-termini containing two cystathionine-β-synthase (CBS) domains. CBS domain secondary structure is highly conserved and consists of two a-helices and three b-strands arranged as b1-a1-b2-b3-a2. ClC CBS domain mutations cause muscle and bone disease and alter ClC gating. However, the precise functional roles of CBS domains and the structural bases by which they regulate ClC function are poorly understood. CLH-3a and CLH-3b are C. elegans ClC anion channel splice variants with strikingly different biophysical properties. Splice variation occurs at cytoplasmic N- and C-termini and includes several amino acids that form a2 of the second CBS domain (CBS2). We demonstrate that interchanging a2 between CLH-3a and CLH-3b interchanges their gating properties. The "R-helix" of ClC proteins forms part of the ion conducting pore and selectivity filter and is connected to the cytoplasmic C-terminus via a short stretch of cytoplasmic amino acids termed the "R-helix linker". C-terminus conformation changes could cause R-helix structural rearrangements via this linker. X-ray structures of three ClC protein cytoplasmic C-termini suggest that a2 of CBS2 and the R-helix linker could be closely apposed and may therefore interact. We found that mutating apposing amino acids in a2 and the R-helix linker of CLH-3b was sufficient to give rise to CLH-3a-like gating. We postulate that the R-helix linker interacts with CBS2 a2, and that this putative interaction provides a pathway by which cytoplasmic C-terminus conformational changes induce conformational changes in membrane domains that in turn modulate ClC function.     

Isolation and Characterization of a High Affinity Peptide Inhibitor of ClC-2 Chloride Channels

The ClC protein family includes voltage-gated chloride channels and chloride/proton exchangers. In eukaryotes, ClC proteins regulate membrane potential of excitable cells, contribute to epithelial transport, and aid in lysosomal acidification. Although structure/function studies of ClC proteins have been aided greatly by the available crystal structures of a bacterial ClC chloride/proton exchanger, the availability of useful pharmacological tools, such as peptide toxin inhibitors, has lagged far behind that of their cation channel counterparts. Here we report the isolation, from Leiurus quinquestriatus hebraeus venom, of a peptide toxin inhibitor of the ClC-2 chloride channel. This toxin, GaTx2, inhibits ClC-2 channels with a voltage-dependent apparent K_D of ∼20 pm, making it the highest affinity inhibitor of any chloride channel. GaTx2 slows ClC-2 activation by increasing the latency to first opening by nearly 8-fold but is unable to inhibit open channels, suggesting that this toxin inhibits channel activation gating. Finally, GaTx2 specifically inhibits ClC-2 channels, showing no inhibitory effect on a battery of other major classes of chloride channels and voltage-gated potassium channels. GaTx2 is the first peptide toxin inhibitor of any ClC protein. The high affinity and specificity displayed by this toxin will make it a very powerful pharmacological tool to probe ClC-2 structure/function.ClC proteins form a family of voltage-gated Cl⁻ channels and Cl⁻/H⁺ exchangers that are found in animals, plants, and bacteria (1). These proteins are expressed on the plasma membrane and some intracellular membranes in both excitable and nonexcitable cells (1, 2). There are nine mammalian members of the ClC family that perform functions as varied as maintenance of membrane potential in neuronal cells (ClC-2) (3), Cl⁻ transport across plasma membranes of epithelial and skeletal muscle cells (ClC-1, ClC-2, and ClC-Ka/b) (1, 4), and participation in lysosomal acidification (ClC-5 and ClC-6) (2). Defects in the genes encoding ClC proteins are linked to a number of diseases including myotonia, epilepsy, Dent''s disease, and Bartter''s syndrome (1–3). It has been suggested recently that ClC-2 may play a role in constipation-associated irritable bowel disease as well as in atherosclerosis (5, 6). Most ClC channels show localized tissue expression; ClC-1, for example, is expressed solely in skeletal muscle, whereas ClC-Ka/b is localized to the kidney. ClC-2, on the other hand, is expressed nearly ubiquitously, suggesting that this channel plays an important, yet largely undefined, physiological role (1, 2).ClC proteins are structurally unrelated to cation channels, with the functional unit being a homodimer (1). ClC channels display two equidistant conductance levels for a single channel opening. In 2002, the crystal structure of a bacterial ClC protein from Salmonella typhimurium was solved, revealing a very complicated membrane topology consisting of 18 α-helical units/subunit in the homodimer, only some of which fully traverse the membrane (7). Examination of the crystal structure revealed no obvious pore, such as is evident in K⁺ channel structures, even though bound Cl⁻ ions were present near the proposed selectivity filter (7, 8). Shortly after the crystal structure was solved, it was shown that the bacterial ClC protein was actually a Cl⁻/H⁺ exchanger and not a channel (9). Comparison of the amino acid sequence of the bacterial ClC protein with that of the eukaryotic ClC channels ClC-0, -1, and -2 revealed only 22, 16, and 19% overall identity, respectively (data not shown). The divergence is largely in the cytoplasmic domains, which are absent in bacterial ClC proteins; sequence identity is much higher in the transmembrane domains.Single-channel gating in ClC proteins is complicated, involving both fast and slow gating processes, which are thought to involve separate regions of the protein (1). Fast gating controls the opening and closing of both protopores independently, operating on the millisecond time scale or faster. Through examination of the crystal structure and subsequent electrophysiological analysis, the fast gating process was revealed to involve a conserved glutamate residue deep within each pore (10). This acidic residue lies near a Cl⁻-binding site and moves slightly to open the pathway in response to changes in membrane voltage and subsequent changes in occupancy of that site, thus providing the link between permeation and gating observed in ClC channels (4). In contrast, slow gating controls both pores simultaneously, operating on the hundreds of milliseconds to seconds time scale. Unlike with fast gating, the regions of the ClC protein involved in slow gating are still unknown, despite the availability of the bacterial ClC crystal structure. It is believed that the dimer interface contributes to slow gating, as well as the long cytoplasmic C-terminal domain, an isolated version of which was recently crystallized (11–13). However, the conformational changes involved in the fast and slow gating processes are still largely unknown. Also, in both ClC-1 and -2, fast and slow gating are linked through an undetermined mechanism (14, 15).Despite the availability of the bacterial ClC protein crystal structure, our understanding of gating mechanisms and structural rearrangements of ClC proteins has lagged behind that of their cation channel counterparts. This is due in large part to a lack of useful pharmacological agents, such as peptide toxins, that may be used as tools. Toxins from venomous animals such as scorpions, snakes, and cone snails have been used for a number of years to define the permeation pathways and gating processes of cation channels (16). However, no peptide toxins have been isolated that inhibit a ClC channel, and only one toxin has been isolated that inhibits any Cl⁻ channel of known molecular identity (17). We recently showed that venom from the scorpion Leiurus quinquestriatus hebraeus contains a peptide component that inhibits the ClC-2 chloride channel (18). Here, we report the isolation of this peptide toxin, its proteomic properties, and primary characteristics of the biophysical mechanism of inhibition.     

Inhibition of ClC-2 Chloride Channels by a Peptide Component or Components of Scorpion Venom

ClC chloride channels play essential roles in membrane excitability and maintenance of osmotic balance. Despite the recent crystallization of two bacterial ClC-like proteins, the gating mechanism for these channels remains unclear. In this study we tested scorpion venom for the presence of novel peptide inhibitors of ClC channels, which might be useful tools for dissecting the mechanisms underlying ClC channel gating. Recently, it has been shown that a peptide component of venom from the scorpion L. quinquestriatus hebraeus inhibits the CFTR chloride channel from the intracellular side. Using two-electrode voltage clamp we studied the effect of scorpion venom on ClC-0, -1, and -2, and found both dose- and voltage-dependent inhibition only of ClC-2. Comparison of voltage-dependence of inhibition by venom to that of known pore blockers revealed opposite voltage dependencies, suggesting different mechanisms of inhibition. Kinetic data show that venom induced slower activation kinetics compared to pre-venom records, suggesting that the active component(s) of venom may function as a gating modifier at ClC-2. Trypsinization abolished the inhibitory activity of venom, suggesting that the component(s) of scorpion venom that inhibits ClC-2 is a peptide.     

Site-directed fluorescence studies of a prokaryotic ClC antiporter

Channels and transporters of the ClC family serve a variety of physiological functions. Understanding of their gating and transport mechanisms remains incomplete, with disagreement over the extent of protein conformational change involved. Using site-directed fluorescence labeling, we probe ClC-ec1, a prokaryotic ClC, for transport-related structural rearrangements. We specifically label cysteines introduced at several positions in the R helix of ClC-ec1 with AlexaFluor 488, an environment-sensitive fluorophore, and demonstrate that the labeled mutants show H+/Cl- transport activity indistinguishable from that of the wild-type protein. At each position that we examined we observe fluorescence changes upon acidification over the same pH range that is known to activate transport. The fluorescence change is also sensitive to Cl- concentration; furthermore, the Cl- and H+ dependencies are coupled as would be expected if the fluorescence change reflected a conformational change required for transport. Together, the results suggest that the changes in fluorescence report protein conformational changes underlying the transport process. Labeled transporters mutated to remove a glutamate critical to proton-coupled chloride transport retain pH-dependent fluorescence changes, suggesting that multiple residues confer pH dependence on the transport mechanism. These results have implications for models of transport and gating in ClC channels and transporters.     

Unique gating properties of C. elegans ClC anion channel splice variants are determined by altered CBS domain conformation and the R-helix linker

All eukaryotic and some prokaryotic ClC anion transport proteins have extensive cytoplasmic C-termini containing two cystathionine-β-synthase (CBS) domains. CBS domain secondary structure is highly conserved and consists of two α-helices and three β-strands arranged as β1-α1-β2-β3-α2. ClC CBS domain mutations cause muscle and bone disease and alter ClC gating. However, the precise functional roles of CBS domains and the structural bases by which they regulate ClC function are poorly understood. CLH-3a and CLH-3b are C. elegans ClC anion channel splice variants with strikingly different biophysical properties. Splice variation occurs at cytoplasmic N- and C-termini and includes several amino acids that form α2 of the second CBS domain (CBS2). We demonstrate that interchanging α2 between CLH-3a and CLH-3b interchanges their gating properties. The “R-helix” of ClC proteins forms part of the ion-conducting pore and selectivity filter and is connected to the cytoplasmic C-terminus via a short stretch of cytoplasmic amino acids termed the “R-helix linker”. C-terminus conformation changes could cause R-helix structural rearrangements via this linker. X-ray structures of three ClC protein cytoplasmic C-termini suggest that α2 of CBS2 and the R-helix linker could be closely apposed and may therefore interact. We found that mutating apposing amino acids in α2 and the R-helix linker of CLH-3b was sufficient to give rise to CLH-3a-LIKE gating. We postulate that the R-helix linker interacts with CBS2 α2, and that this putative interaction provides a pathway by which cytoplasmic C-terminus conformational changes induce conformational changes in membrane domains that in turn modulate ClC function.Key words: ClC channel, chloride channel, homology model     

Secondary Water Pore Formation for Proton Transport in a ClC Exchanger Revealed by an Atomistic Molecular-Dynamics Simulation

Several prokaryotic ClC proteins have been demonstrated to function as exchangers that transport both chloride ions and protons simultaneously in opposite directions. However, the path of the proton through the ClC exchanger, and how the protein brings about the coupled movement of both ions are still unknown. In this work, we use an atomistic molecular dynamics (MD) simulation to demonstrate that a previously unknown secondary water pore is formed inside an Escherichia coli ClC exchanger. The secondary water pore is bifurcated from the chloride ion pathway at E148. From the systematic simulations, we determined that the glutamate residue exposed to the intracellular solution, E203, plays an important role as a trigger for the formation of the secondary water pore, and that the highly conserved tyrosine residue Y445 functions as a barrier that separates the proton from the chloride ion pathways. Based on our simulation results, we conclude that protons in the ClC exchanger are conducted via a water network through the secondary water pore, and we propose a new mechanism for the coupled transport of chloride ions and protons. It has been reported that several members of ClC proteins are not just channels that simply transport chloride ions across lipid bilayers; rather, they are exchangers that transport both the chloride ion and proton in opposite directions. However, the ion transit pathways and the mechanism of the coupled movement of these two ions have not yet been unveiled. In this article, we report a new finding (to our knowledge) of a water pore inside a prokaryotic ClC protein as revealed by computer simulation. This water pore is bifurcated from the putative chloride ion, and water molecules inside the new pore connect two glutamate residues that are known to be key residues for proton transport. On the basis of our simulation results, we conclude that the water wire that is formed inside the newly found pore acts as a proton pathway, which enables us to resolve many problems that could not be addressed by previous experimental studies.     

Rescue of DeltaF508-CFTR trafficking and gating in human cystic fibrosis airway primary cultures by small molecules

Cystic fibrosis (CF) is a fatal genetic disease caused by mutations in cftr, a gene encoding a PKA-regulated Cl(-) channel. The most common mutation results in a deletion of phenylalanine at position 508 (DeltaF508-CFTR) that impairs protein folding, trafficking, and channel gating in epithelial cells. In the airway, these defects alter salt and fluid transport, leading to chronic infection, inflammation, and loss of lung function. There are no drugs that specifically target mutant CFTR, and optimal treatment of CF may require repair of both the folding and gating defects. Here, we describe two classes of novel, potent small molecules identified from screening compound libraries that restore the function of DeltaF508-CFTR in both recombinant cells and cultures of human bronchial epithelia isolated from CF patients. The first class partially corrects the trafficking defect by facilitating exit from the endoplasmic reticulum and restores DeltaF508-CFTR-mediated Cl(-) transport to more than 10% of that observed in non-CF human bronchial epithelial cultures, a level expected to result in a clinical benefit in CF patients. The second class of compounds potentiates cAMP-mediated gating of DeltaF508-CFTR and achieves single-channel activity similar to wild-type CFTR. The CFTR-activating effects of the two mechanisms are additive and support the rationale of a drug discovery strategy based on rescue of the basic genetic defect responsible for CF.     

High-level expression, functional reconstitution, and quaternary structure of a prokaryotic ClC-type chloride channel.

ClC-type anion-selective channels are widespread throughout eukaryotic organisms. BLAST homology searches reveal that many microbial genomes also contain members of the ClC family. An Escherichia coli-derived ClC Cl(-) channel homologue, "EriC," the product of the yadQ gene, was overexpressed in E. coli and purified in milligram quantities in a single-step procedure. Reconstitution of purified EriC into liposomes confers on these membranes permeability to anions with selectivity similar to that observed electrophysiologically in mammalian ClC channels. Cross-linking studies argue that EriC is a homodimer in both detergent micelles and reconstituted liposomes, a conclusion corroborated by gel filtration and analytical sedimentation experiments.     

Localization of a putative ClC chloride channel in spinach chloroplasts

Seven genes seem to encode for putative ClC chloride channels (AtClC-a to AtClC-g) in Arabidopsis thaliana. Their function and localization is still largely unknown. AtClC-f shares considerable sequence similarity with putative ClC channel proteins from Synechocystis, considered to represent the precursor of chloroplasts. We show by biochemical and mass spectrometry analysis that ClC-f is located in the outer envelope membrane of spinach chloroplasts. Consistent with the plastidial localization of ClC-f, p-chlorophenoxy-acetic acid (CPA) reduces photosynthetic activity and the protein is expressed in etioplasts and chloroplasts but not in root tissue. These findings may represent a step toward the molecular identification of ion channel activities in chloroplast membranes.     

Identification of Regulatory Phosphorylation Sites in a Cell Volume– and Ste20 Kinase–dependent ClC Anion Channel

Changes in phosphorylation regulate the activity of various ClC anion transport proteins. However, the physiological context under which such regulation occurs and the signaling cascades that mediate phosphorylation are poorly understood. We have exploited the genetic model organism Caenorhabditis elegans to characterize ClC regulatory mechanisms and signaling networks. CLH-3b is a ClC anion channel that is expressed in the worm oocyte and excretory cell. Channel activation occurs in response to oocyte meiotic maturation and swelling via serine/threonine dephosphorylation mediated by the type I phosphatases GLC-7α and GLC-7β. A Ste20 kinase, germinal center kinase (GCK)-3, binds to the cytoplasmic C terminus of CLH-3b and inhibits channel activity in a phosphorylation-dependent manner. Analysis of hyperpolarization-induced activation kinetics suggests that phosphorylation may inhibit the ClC fast gating mechanism. GCK-3 is an ortholog of mammalian SPAK and OSR1, kinases that bind to, phosphorylate, and regulate the cell volume–dependent activity of mammalian cation-Cl⁻ cotransporters. Using mass spectrometry and patch clamp electrophysiology, we demonstrate here that CLH-3b is a target of regulatory phosphorylation. Concomitant phosphorylation of S742 and S747, which are located 70 and 75 amino acids downstream from the GCK-3 binding site, are required for kinase-mediated channel inhibition. In contrast, swelling-induced channel activation occurs with dephosphorylation of S747 alone. Replacement of both S742 and S747 with glutamate gives rise to kinase- and swelling-insensitive channels that exhibit activity and biophysical properties similar to those of wild-type CLH-3b inhibited by GCK-3. Our studies provide novel insights into ClC regulation and mechanisms of cell volume signaling, and provide the foundation for studies aimed at defining how conformational changes in the cytoplasmic C terminus alter ClC gating and function in response to intracellular signaling events.     

Conduction mechanisms of chloride ions in ClC-type channels

The conduction properties of ClC-0 and ClC-1 chloride channels are examined using electrostatic calculations and three-dimensional Brownian dynamics simulations. We create an open-state configuration of the prokaryotic ClC Cl(-) channel using its known crystallographic structure as a basis. Two residues that are occluding the channel are slowly pushed outward with molecular dynamics to create a continuous ion-conducting path with the minimum radius of 2.5 A. Then, retaining the same pore shape, the prokaryotic ClC channel is converted to either ClC-0 or ClC-1 by replacing all the nonconserved dipole-containing and charged amino acid residues. Employing open-state ClC-0 and ClC-1 channel models, current-voltage curves consistent with experimental measurements are obtained. We find that conduction in these pores involves three ions. We locate the binding sites, as well as pinpointing the rate-limiting steps in conduction, and make testable predictions about how the single channel current across ClC-0 and ClC-1 will vary as the ionic concentrations are increased. Finally, we demonstrate that a ClC-0 homology model created from an alternative sequence alignment fails to replicate any of the experimental observations.     

From stones to bones: the biology of ClC chloride channels

Chloride (Cl(-)) is the most abundant extracellular anion in multicellular organisms. Passive movement of Cl(-) through membrane ion channels enables several cellular and physiological processes including transepithelial salt transport, electrical excitability, cell volume regulation and acidification of internal and external compartments. One family of proteins mediating Cl(-) permeability, the ClC channels, has emerged as important for all of these biological processes. The importance of ClC channels has in part been realized through studies of inherited human diseases and genetically engineered mice that display a wide range of phenotypes from kidney stones to petrified bones. These recent findings have demonstrated many eclectic functions of ClC channels and have placed Cl(-) channels in the physiological limelight.     

Linker-gating ring complex as passive spring and Ca(2+)-dependent machine for a voltage- and Ca(2+)-activated potassium channel

Ion channels are proteins that control the flux of ions across cell membranes by opening and closing (gating) their pores. It has been proposed that channels gated by internal agonists have an intracellular gating ring that extracts free energy from agonist binding to open the gates using linkers that directly connect the gating ring to the gates. Here we find for a voltage- and Ca(2+)-activated K+ (BK) channel that shortening the linkers increases channel activity and lengthening the linkers decreases channel activity, both in the presence and absence of intracellular Ca2+. These observations are consistent with a mechanical model in which the linker-gating ring complex forms a passive spring that applies force to the gates in the absence of Ca2+ to modulate the voltage-dependent gating. Adding Ca2+ then changes the force to further activate the channel. Both the passive and Ca(2+)-induced forces contribute to the gating of the channel.     

Ion-binding properties of the ClC chloride selectivity filter

The ClC channels are members of a large protein family of chloride (Cl-) channels and secondary active Cl- transporters. Despite their diverse functions, the transmembrane architecture within the family is conserved. Here we present a crystallographic study on the ion-binding properties of the ClC selectivity filter in the close homolog from Escherichia coli (EcClC). The ClC selectivity filter contains three ion-binding sites that bridge the extra- and intracellular solutions. The sites bind Cl- ions with mM affinity. Despite their close proximity within the filter, the three sites can be occupied simultaneously. The ion-binding properties are found conserved from the bacterial transporter EcClC to the human Cl- channel ClC-1, suggesting a close functional link between ion permeation in the channels and active transport in the transporters. In resemblance to K+ channels, ions permeate the ClC channel in a single file, with mutual repulsion between the ions fostering rapid conduction.     

Structure-function discrimination of the N- and C- catalytic domains of human angiotensin-converting enzyme: implications for Cl- activation and peptide hydrolysis mechanisms

Human somatic angiotensin I-converting enzyme (sACE) has two active sites present in two sequence homologous protein domains (ACE_N and ACE_C) possessing several biochemical features that differentiate the two active sites (i.e. chloride ion activation). Based on the recently solved X-ray structure of testis angiotensin-converting enzyme (tACE), the 3D structure of ACE_N was modeled. Electrostatic potential calculations reveal that the ACE_N binding groove is significantly more positively charged than the ACE_C, which provides a first rationalization for their functional discrimination. The chloride ion pore for Cl2 (one of the two chloride ions revealed in the X-ray structure of tACE) that connects the external solution with the inner part of the protein was identified on the basis of an extended network of water molecules. Comparison of ACE_C with the X-ray structure of the prokaryotic ClC Cl(-) channel from Salmonella enterica serovar typhimurium demonstrates a common molecular basis of anion selectivity. The critical role for Cl2 as an ionic switch is emphasized. Sequence and structural comparison between ACE_N and ACE_C and of other proteins of the gluzincin family highlights key residues that could be responsible for the peptide hydrolysis mechanism. Currently available mutational and substrate hydrolysis data for both domains are evaluated and are consistent with the predicted model.