Nucleotide recognition by the cytoplasmic domain of the human chloride transporter ClC-5

The ubiquitous CBS domains, which are found as part of cytoplasmic domains in the ClC family of chloride channels and transporters, have previously been identified as building blocks for regulatory nucleotide-binding sites. Here we report the structures of the cytoplasmic domain of the human transporter ClC-5 in complex with ATP and ADP. The nucleotides bind to a specific site in the protein. As determined by equilibrium dialysis, the affinities for ATP, ADP and AMP are in the high micromolar range. Point mutations that interfere with nucleotide binding change the transport behavior of a ClC-5 mutant expressed in Xenopus laevis oocytes. Our results establish the structural and energetic basis for the interaction of ClC-5 with nucleotides and provide a framework for future investigations.     

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.     

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     

Two splice variants derived from a Drosophila melanogaster candidate ClC gene generate ClC-2-type Cl- channels

Members of the ClC family of membrane proteins have been found in a variety of species and they can function as Cl- channels or Cl-/H+ antiporters. Three potential ClC genes are present in the Drosophila melanogaster genome. Only one of them shows homology with a branch of the mammalian ClC genes that encode plasma membrane Cl- channels. The remaining two are close to mammalian homologues coding for intracellular ClC proteins. Using RT-PCR we have identified two splice variants showing highest homology (41% residue identity) to the mammalian ClC-2 chloride channel. One splice variant (DmClC-2S) is expressed in the fly head and body and an additional, larger variant (DmClC-2L) is only present in the head. Both putative Drosophila channels conserve key features of the ClC channels cloned so far, including residues conforming the selectivity filter and C-terminus CBS domains. The splice variants differ in a stretch of 127 aa at the intracellular C-terminal portion separating cystathionate beta synthase (CBS) domains. Expression of either Drosophila ClC-2 variant in HEK-293 cells generated inwardly rectifying Cl- currents with similar activation and deactivation characteristics. There was great similarity in functional characteristics between DmClC-2 variants and their mammalian counterpart, save for slower opening kinetics and faster closing rate. As CBS domains are believed to be sites of regulation of channel gating and trafficking, it is suggested that the extra amino acids present between CBS domains in DmClC-2L might endow the channel with a differential response to signals present in the fly cells where it is expressed.     

The structure of the cytoplasmic domain of the chloride channel ClC-Ka reveals a conserved interaction interface

The cytoplasmic domains of ClC chloride channels and transporters are ubiquitously found in eukaryotic family members and have been suggested to be involved in the regulation of ion transport. All cytoplasmic ClC domains share a conserved scaffold that contains a pair of CBS motifs. Here we describe the structure of the cytoplasmic component of the human chloride channel ClC-Ka at 1.6 A resolution. The structure reveals a dimeric organization of the domain that is unusual for CBS motif containing proteins. Using a biochemical approach combining mutagenesis, crosslinking, and analytical ultracentrifugation, we demonstrate that the interaction interface is preserved in solution and that the distantly related channel ClC-0 likely exhibits a similar structural organization. Our results reveal a conserved interaction interface that relates the cytoplasmic domains of ClC proteins and establish a structural relationship that is likely general for this important family of transport proteins.     

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.     

Evaluation of the membrane-spanning domain of ClC-2

The ClC family of chloride channels and transporters includes several members in which mutations have been associated with human disease. An understanding of the structure-function relationships of these proteins is essential for defining the molecular mechanisms underlying pathogenesis. To date, the X-ray crystal structures of prokaryotic ClC transporter proteins have been used to model the membrane domains of eukaryotic ClC channel-forming proteins. Clearly, the fidelity of these models must be evaluated empirically. In the present study, biochemical tools were used to define the membrane domain boundaries of the eukaryotic protein, ClC-2, a chloride channel mutated in cases of idiopathic epilepsy. The membrane domain boundaries of purified ClC-2 and accessible cysteine residues were determined after its functional reconstitution into proteoliposomes, labelling using a thiol reagent and proteolytic digestion. Subsequently, the lipid-embedded and soluble fragments generated by trypsin-mediated proteolysis were studied by MS and coverage of approx. 71% of the full-length protein was determined. Analysis of these results revealed that the membrane-delimited boundaries of the N- and C-termini of ClC-2 and the position of several extramembrane loops determined by these methods are largely similar to those predicted on the basis of the prokaryotic protein [ecClC (Escherichia coli ClC)] structures. These studies provide direct biochemical evidence supporting the relevance of the prokaryotic ClC protein structures towards understanding the structure of mammalian ClC channel-forming proteins.     

The muscle chloride channel ClC-1 is not directly regulated by intracellular ATP

ClC-1 belongs to the gene family of CLC Cl(-) channels and Cl(-)/H(+) antiporters. It is the major skeletal muscle chloride channel and is mutated in dominant and recessive myotonia. In addition to the membrane-embedded part, all mammalian CLC proteins possess a large cytoplasmic C-terminal domain that bears two so-called CBS (from cystathionine-beta-synthase) domains. Several studies indicate that these domains might be involved in nucleotide binding and regulation. In particular, Bennetts et al. (J. Biol. Chem. 2005. 280:32452-32458) reported that the voltage dependence of hClC-1 expressed in HEK cells is regulated by intracellular ATP and other nucleotides. Moreover, very recently, Bennetts et al. (J. Biol. Chem. 2007. 282:32780-32791) and Tseng et al. (J. Gen. Physiol. 2007. 130:217-221) reported that the ATP effect was enhanced by intracellular acidification. Here, we show that in striking contrast with these findings, human ClC-1, expressed in Xenopus oocytes and studied with the inside-out configuration of the patch-clamp technique, is completely insensitive to intracellular ATP at concentrations up to 10 mM, at neutral pH (pH 7.3) as well as at slightly acidic pH (pH 6.2). These results have implications for a general understanding of nucleotide regulation of CLC proteins and for the physiological role of ClC-1 in muscle excitation.     

The role of the carboxyl terminus in ClC chloride channel function

The human muscle chloride channel ClC-1 has a 398-amino acid carboxyl-terminal domain that resides in the cytoplasm and contains two CBS (cystathionine-beta-synthase) domains. To examine the role of this region, we studied various carboxyl-terminal truncations by heterologous expression in mammalian cells, whole-cell patch clamp recording, and confocal imaging. Channel constructs lacking parts of the distal CBS domain, CBS2, did not produce functional channels, whereas deletion of CBS1 was tolerated. ClC channels are dimeric proteins with two ion conduction pathways (protopores). In heterodimeric channels consisting of one wild type subunit and one subunit in which the carboxyl terminus was completely deleted, only the wild type protopore was functional, indicating that the carboxyl terminus supports the function of the protopore. All carboxyl-terminal-truncated mutant channels fused to yellow fluorescent protein were translated and the majority inserted into the plasma membrane as revealed by confocal microscopy. Fusion proteins of cyan fluorescent protein linked to various fragments of the carboxyl terminus formed soluble proteins that could be redistributed to the surface membrane through binding to certain truncated channel subunits. Stable binding only occurs between carboxyl-terminal fragments of a single subunit, not between carboxyl termini of different subunits and not between carboxyl-terminal and transmembrane domains. However, an interaction with transmembrane domains can modify the binding properties of particular carboxyl-terminal proteins. Our results demonstrate that the carboxyl terminus of ClC-1 is not necessary for intracellular trafficking but is critical for channel function. Carboxyl termini fold independently and modify individual protopores of the double-barreled channel.     

Cytoplasmic ATP-sensing domains regulate gating of skeletal muscle ClC-1 chloride channels

ClC proteins are a family of chloride channels and transporters that are found in a wide variety of prokaryotic and eukaryotic cell types. The mammalian voltage-gated chloride channel ClC-1 is important for controlling the electrical excitability of skeletal muscle. Reduced excitability of muscle cells during metabolic stress can protect cells from metabolic exhaustion and is thought to be a major factor in fatigue. Here we identify a novel mechanism linking excitability to metabolic state by showing that ClC-1 channels are modulated by ATP. The high concentration of ATP in resting muscle effectively inhibits ClC-1 activity by shifting the voltage gating to more positive potentials. ADP and AMP had similar effects to ATP, but IMP had no effect, indicating that the inhibition of ClC-1 would only be relieved under anaerobic conditions such as intense muscle activity or ischemia, when depleted ATP accumulates as IMP. The resulting increase in ClC-1 activity under these conditions would reduce muscle excitability, thus contributing to fatigue. We show further that the modulation by ATP is mediated by cystathionine beta-synthase-related domains in the cytoplasmic C terminus of ClC-1. This defines a function for these domains as gating-modulatory domains sensitive to intracellular ligands, such as nucleotides, a function that is likely to be conserved in other ClC proteins.     

Multimeric structure of ClC-1 chloride channel revealed by mutations in dominant myotonia congenita (Thomsen).

Voltage-gated ClC chloride channels play important roles in cell volume regulation, control of muscle excitability, and probably transepithelial transport. ClC channels can be functionally expressed without other subunits, but it is unknown whether they function as monomers. We now exploit the properties of human mutations in the muscle chloride channel, ClC-1, to explore its multimeric structure. This is based on analysis of the dominant negative effects of ClC-1 mutations causing myotonia congenita (MC, Thomsen's disease), including a newly identified mutation (P480L) in Thomsen's own family. In a co-expression assay, Thomsen's mutation dramatically inhibits normal ClC-1 function. A mutation found in Canadian MC families (G230E) has a less pronounced dominant negative effect, which can be explained by functional WT/G230E heterooligomeric channels with altered kinetics and selectivity. Analysis of both mutants shows independently that ClC-1 functions as a homooligomer with most likely four subunits.     

The chloride channel ClC-4 co-localizes with cystic fibrosis transmembrane conductance regulator and may mediate chloride flux across the apical membrane of intestinal epithelia.

Cystic fibrosis (CF) causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) lead to mislocalization of CFTR protein from the brush border membrane of epithelial tissues and/or its dysfunction as a chloride channel. In initial reports, it was proposed that certain channels from the ClC family of chloride channels may provide compensatory or alternative pathways for epithelial chloride secretion in tissues from cystic fibrosis patients. In the present work, we provide the first evidence that ClC-4 protein is functionally expressed on the surface of the intestinal epithelium and hence, is appropriately localized to act as a therapeutic target in this CF-affected tissue. We show using confocal and electron microscopy that ClC-4 co-localizes with CFTR in the brush border membrane of the epithelium lining intestinal crypts in mouse and human tissues. In Caco-2 cells, a cell line thought to model human enterocytes, ClC-4 protein is expressed on the cell surface and also partially co-localizes with EEA1 and transferrin, marker molecules of early and recycling endosomes, respectively. Hence, like CFTR, ClC-4 may cycle between the plasma membrane and endosomal compartment. Furthermore, we show that ClC-4 functions as a chloride channel on the surface of these epithelial cells as antisense ClC-4 cDNA expression reduced the amplitude of endogenous chloride currents by 50%. These studies provide the first evidence that ClC-4 is endogenously expressed and may be functional in the brush border membrane of enterocytes and hence should be considered as a candidate channel to provide an alternative pathway for chloride secretion in the gastrointestinal tract of CF patients.     

The PDZ-binding chloride channel ClC-3B localizes to the Golgi and associates with cystic fibrosis transmembrane conductance regulator-interacting PDZ proteins

ClC chloride channels are widely distributed in organisms across the evolutionary spectrum, and members of the mammalian family play crucial roles in cellular function and are mutated in several human diseases (Jentsch, T. J., Stein, V., Weinreich, F., and Zdebik, A. A. (2002) Physiol. Rev. 82, 503-568). Within the ClC-3, -4, -5 branch of the family that are intracellular channels, two alternatively spliced ClC-3 isoforms were recognized recently (Ogura, T., Furukawa, T., Toyozaki, T., Yamada, K., Zheng, Y. J., Katayama, Y., Nakaya, H., and Inagaki, N. (2002) FASEB J. 16, 863-865). ClC-3A resides in late endosomes where it serves as an anion shunt during acidification. We show here that the ClC-3B PDZ-binding isoform resides in the Golgi where it co-localizes with a small amount of the other known PDZ-binding chloride channel, CFTR (cystic fibrosis transmembrane conductance regulator). Both channel proteins bind the Golgi PDZ protein, GOPC (Golgi-associated PDZ and coiled-coil motif-containing protein). Interestingly, however, when overexpressed, GOPC, which is thought to influence traffic in the endocytic/secretory pathway, causes a large reduction in the amounts of both channels, probably by leading them to the degradative end of this pathway. ClC-3B as well as CFTR also binds EBP50 (ERM-binding phosphoprotein 50) and PDZK1, which are concentrated at the plasma membrane. However, only PDZK1 was found to promote interaction between the two channels, perhaps because they were able to bind to two different PDZ domains in PDZK1. Thus while small portions of the populations of ClC-3B and CFTR may associate and co-localize, the bulk of the two populations reside in different organelles of cells where they are expressed heterologously or endogenously, and therefore their cellular functions are likely to be distinct and not primarily related.     

The poststructural festivities begin

ClC chloride channels orchestrate the movement of chloride necessary for proper neuronal, muscular, cardiovascular, and epithelial function. In this issue of Neuron, Jentsch, Pusch, and colleagues use the structure of a bacterial ClC homolog to guide a mutagenic analysis of inhibitor binding to ClC-0, ClC-1, and ClC-2.     

Intracellular localization of ClC chloride channels and their ability to form hetero-oligomers

ClC chloride channels (ClCs) can be classified into two groups in terms of their cellular localizations: ClCs present in the plasma membranes and those residing in intracellular organelles. Members of the latter group, including ClC-3, ClC-4, ClC-5, ClC-6, and ClC-7, are often co-expressed in a variety of cell types in many organs. Although the localization of individual channels within cells has been investigated, the degree of overlap between the locations of different ClCs in the same cell has not been clarified. To address this question, different combinations of ClCs, engineered to encode specific epitope tags (FLAG or HA), were either transiently or stably transfected into HEK293 cells, and we then compared the intracellular localization of the expressed channel proteins by immunofluorescence microscopy. Immunofluorescence images of the alternatively labeled channels clearly showed significant co-localization between all pair-wise combinations of ClCs. In particular, ClC-3, ClC-4, and ClC-5 showed a high degree of co-localization. As a significant degree of co-localization between ClCs was observed, we used co-immunoprecipitation to evaluate oligomer formation, and found that each ClC tested could form homo-oligomers, and that any pair-wise combination of ClC-3, ClC-4, and ClC-5 could also form hetero-oligomers. Neither ClC-6 nor ClC-7 was co-precipitated with any other channel protein. These results suggest that within cells ClC-3, ClC-4, and ClC-5 may have combinatorial functions, whereas ClC-6 and ClC-7 are more likely to function as homo-oligomers.     

Crystal structure of the cytoplasmic domain of the chloride channel ClC-0

Ion channels are frequently organized in a modular fashion and consist of a membrane-embedded pore domain and a soluble regulatory domain. A similar organization is found for the ClC family of Cl- channels and transporters. Here, we describe the crystal structure of the cytoplasmic domain of ClC-0, the voltage-dependent Cl- channel from T. marmorata. The structure contains a folded core of two tightly interacting cystathionine beta-synthetase (CBS) subdomains. The two subdomains are connected by a 96 residue mobile linker that is disordered in the crystals. As revealed by analytical ultracentrifugation, the domains form dimers, thereby most likely extending the 2-fold symmetry of the transmembrane pore. The structure provides insight into the organization of the cytoplasmic domains within the ClC family and establishes a framework for guiding future investigations on regulatory mechanisms.     

Biophysical properties of ClC-3 differentiate it from swelling-activated chloride channels in Chinese hamster ovary-K1 cells

ClC-3 is a highly conserved voltage-gated chloride channel, which together with ClC-4 and ClC-5 belongs to one subfamily of the larger group of ClC chloride channels. Whereas ClC-5 is localized intracellularly, ClC-3 has been reported to be a swelling-activated plasma membrane channel. However, recent studies have shown that native ClC-3 in hepatocytes is primarily intracellular. Therefore, we reexamined the properties of ClC-3 in a mammalian cell expression system and compared them with the properties of endogenous swelling-activated channels. Chinese hamster ovary (CHO)-K1 cells were transiently transfected with rat ClC-3. The resulting chloride currents were Cl(-) > I(-) selective, showed extreme outward rectification, and lacked inactivation at positive voltages. In addition, they were insensitive to the chloride channel blockers, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and were not inhibited by phorbol esters or activated by osmotic swelling. These properties are identical to those of ClC-5 but differ from those previously attributed to ClC-3. In contrast, nontransfected CHO-K1 cells displayed an endogenous swelling-activated chloride current, which was weakly outward rectifying, inactivated at positive voltages, sensitive to NPPB and DIDS, and inhibited by phorbol esters. These properties are identical to those previously attributed to ClC-3. Therefore, we conclude that when expressed in CHO-K1 cells, ClC-3 is an extremely outward rectifying channel with similar properties to ClC-5 and is neither activated by cell swelling nor identical to the endogenous swelling-activated channel. These data suggest that ClC-3 cannot be responsible for the swelling-activated chloride channel under all circumstances.     

A structural perspective on ClC channel and transporter function

The ClC chloride channels and transporters constitute a large family of membrane proteins that is involved in a variety of physiological processes. All members share a conserved molecular architecture that consists of a complex transmembrane transport domain followed by a cytoplasmic domain. Despite the strong conservation, the family shows an unusually broad variety of functional behaviors as some members work as gated chloride channels and others as secondary active chloride transporters. The conservation in the structure and the functional resemblance of gating and coupled transport suggests a strong mechanistic relationship between these seemingly contradictory transport modes. The cytoplasmic domains constitute putative regulatory components that are ubiquitous in eukaryotic ClC family members and that in certain cases interact with nucleotides thus linking ion transport to nucleotide sensing by yet unknown mechanisms.     

Overexpression of CLC-3 in HEK293T cells yields novel currents that are pH dependent

ClC-3 is a member of the ClC family of anion channels/transporters. Recently, the closely related proteins ClC-4 and ClC-5 were shown to be Cl(-)/H(+) antiporters (39, 44). The function of ClC-3 has been controversial. We studied anion currents in HEK293T cells expressing wild-type or mutant ClC-3. The basic biophysical properties of ClC-3 currents were very similar to those of ClC-4 and ClC-5, and distinct from those of the swelling-activated anion channel. ClC-3 expression induced currents with time-dependent activation that rectified sharply in the outward direction. The reversal potential of the current shifted by -48.3 +/- 2.5 mV per 10-fold (decade) change in extracellular Cl(-) concentration, which did not conform to the behavior of an anion-selective channel based upon the Nernst equation, which predicts a -58.4 mV/decade shift at 22 degrees C. Manipulation of extracellular pH (6.35-8.2) altered reversal potential by 10.2 +/- 3.0 mV/decade, suggesting that ClC-3 currents were coupled to proton movement. Mutation of a specific glutamate residue (E224A) changed voltage dependence in a manner similar to that observed in other ClC Cl(-)/H(+) antiporters. Mutant currents exhibited Nernstian changes in reversal potential in response to altered extracellular Cl(-) concentration that averaged -60 +/- 3.4 mV/decade and were pH independent. Thus ClC-3 overexpression induced a pH-sensitive conductance in HEK293T cells that is biophysically similar to ClC-4 and ClC-5.