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.     

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     

The fast gating mechanism in ClC-0 channels

We investigate and then modify the hypothesis that a glutamate side chain acts as the fast gate in ClC-0 channels. We first create a putative open-state configuration of the prokaryotic ClC Cl- channel using its crystallographic structure as a basis. Then, retaining the same pore shape, the prokaryotic ClC channel is converted to ClC-0 by replacing all the nonconserved polar and charged residues. Using this open-state channel model, we carry out molecular dynamics simulations to study how the glutamate side chain can move between open and closed configurations. When the side chain extends toward the extracellular end of the channel, it presents an electrostatic barrier to Cl- conduction. However, external Cl- ions can push the side chain into a more central position where, pressed against the channel wall, it does not impede the motion of Cl- ions. Additionally, a proton from a low-pH external solution can neutralize the extended glutamate side chain, which also removes the barrier to conduction. Finally, we use Brownian dynamics simulations to demonstrate the influence of membrane potential and external Cl- concentration on channel open probability.     

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.     

Bead-like passage of chloride ions through ClC chloride channels

The ClC chloride channels control the ionic composition of the cytoplasm and the volume of cells, and regulate electrical excitability. Recently, it has been proposed that prokaryotic ClC channels are H+-Cl- exchange transporter. Although X-ray and molecular dynamics (MD) studies of bacterial ClC channels have investigated the filter open-close and ion permeation mechanism of channels, details have remained unclear. We performed MD simulations of ClC channels involving H+, Na+, K+, or H3O+ in the intracellular region to elucidate the open-close mechanism, and to clarify the role of H+ ion an H+-Cl- exchange transporter. Our simulations revealed that H+ and Na+ caused channel opening and the passage of Cl- ions. Na+ induced a bead-like string of Cl- -Na+-Cl--Na+-Cl- ions to form and permeate through ClC channels to the intracellular side with the widening of the channel pathway.     

Tn5530 from Burkholderia cepacia strain 2a encodes a chloride channel protein essential for the catabolism of 2,4-dichlorophenoxyacetic acid

Chloride channel proteins (ClC) are found in living systems where they transport chloride ions across cell membranes. Recently, the structure/function of two prokaryotic ClC has been determined but little is known about the role of these proteins in the microbial metabolism of chlorinated compounds. Here we show that transposon Tn5530 from Burkholderia cepacia strain 2a encodes a ClC protein (BcClC) which is responsible for expelling Cl(-) ions generated during the catabolism of 2,4-dichlorophenoxyacetic acid (a chlorinated herbicide). We found that BcClC has the ability to transport Cl(-) ions across reconstituted proteoliposome membranes. We created two mutants in which the intrachannel glutamate residue of the protein, known to be responsible for opening and closing the channel (i.e. gating), was changed in order to create constitutively open and closed forms. We observed that cells carrying the closed-channel protein accumulated Cl(-) ions intracellularly leading to a decrease in intracellular pH, cell stasis and death. Further, we established that BcClC has the same gating mechanism as that reported for the ClC protein from Salmonella typhimurium. Our results show that the physiological role of ClC is to maintain cellular homeostasis which can be impaired by the catabolism of chlorinated compounds.     

Probing the pore of ClC-0 by substituted cysteine accessibility method using methane thiosulfonate reagents

ClC channels are a family of protein molecules containing two ion-permeation pores. Although these transmembrane proteins are important for a variety of physiological functions, their molecular operations are only superficially understood. High-resolution X-ray crystallography techniques have recently revealed the structures of two bacterial ClC channels, but whether vertebrate ClC channel pores are similar to those of bacterial homologues is not clear. To study the pore architecture of the Torpedo ClC-0 channel, we employed the substituted-cysteine-accessibility method (SCAM) and used charged methane thiosulfonate (MTS) compounds to modify the introduced cysteine. Several conclusions were derived from this approach. First, the MTS modification pattern from Y512C to E526C in ClC-0, which corresponds to residues forming helix R in bacterial ClC channels, is indeed consistent with the suggested helical structure. Second, the ClC-0 pore is more accessible to the negatively charged than to the positively charged MTS compound, a pore property that is regulated by the intrinsic electrostatic potential in the pore. Finally, attempts to modify the introduced cysteine at positions intracellular to the selectivity filter did not result in larger MTS modification rates for the open-state channel, suggesting that the fast gate of ClC-0 cannot be located at a position intracellular to the Cl- selectivity filter. Thus, the proposal that the glutamate side chain is the fast gate of the channel is applicable to ClC-0, revealing a structural and functional conservation of ClC channels between bacterial and vertebrate species.     

Rapid recycling of ClC‐2 chloride channels between plasma membrane and endosomes: Role of a tyrosine endocytosis motif in surface retrieval

ClC‐2 chloride channel is present in the brain and some transporting epithelia where its function is poorly understood. We have now demonstrated that the surface channels are rapidly internalised and approximately the 70% of the surface membrane protein recycles after 4‐ to 8‐min internalisation. Endocytosis of ClC‐2 was dependent upon tyrosine 179 located within an endocytic motif. Rapid recycling accompanied by an even faster internalisation could account for the abundant presence of ClC‐2 in intracellular membranous structures. At least a proportion of ClC‐2 resides in lipid rafts. Use of β‐cyclodextrin led to an increase in cell surface channel, but, surprisingly, a decrease in functionally active channels. We suggest that ClC‐2 requires residing in β‐cyclodextrin sensitive clusters with other molecules in order to remain active. Regulation of ClC‐2 trafficking to and within the membrane could be a means of modulating its activity. J. Cell. Physiol. 221: 650–657, 2009. © 2009 Wiley‐Liss, Inc.     

The cytoplasmic domain of the chloride channel ClC-0: structural and dynamic characterization of flexible regions

Eukaryotic members of the ClC family of chloride channels and transporters are composed of a transmembrane ion transport domain followed by a cytoplasmic domain, which is believed to be involved in the modulation of ClC function. In some family members this putative regulatory domain contains next to a well-folded structured part, long sequence stretches with low sequence complexity. These regions, a 96 residue long linker connecting two structured sub-domains, and 35 residues on the C teminus of the domain were found disordered in a recent crystal structure of this domain in ClC-0. Both regions have a large influence on the modulation of channel function in closely related family members. Here we describe a NMR study to characterize the structural and dynamic properties of these putatively unstructured stretches. Our study reveals that the two regions indeed show large conformational flexibility with dynamics on the nanosecond timescale. However, small islands of secondary structure are found interdispersed between the unfolded regions. This study characterizes for the first time the biophysical properties of these protein segments, which may become important for the understanding of novel regulatory mechanisms within the ClC family.     

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.     

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.     

The C-terminal C1 cassette of the N -methyl-D-aspartate receptor 1 subunit contains a bi-partite nuclear localization sequence

The N -methyl-D-aspartate receptor (NMDAR) is a multimeric transmembrane protein composed of at least two subunits. One subunit, NR1, is derived from a single gene and can be subdivided into three regions: the N-terminal extracellular domain, the transmembrane regions, and the C-terminal intracellular domain. The N-terminal domain is responsible for Mg2+ metal ion binding and channel activity, while the transmembrane domains are important for ion channel formation. The intracellular C-terminal domain is involved in regulating receptor activity and subcellular localization. Our recent experiments indicated that the intracellular C-terminal domain, when expressed independently, localizes almost exclusively in the nucleus. An examination of the amino acid sequence reveals the presence of a putative nuclear localization sequence (NLS) in the C1 cassette of the NR1 intracellular C-terminus. Using an expression vector designed to test whether a putative NLS sequence is a valid, functional NLS, we have demonstrated that a bi-partite NLS does in fact exist within the NR1-1 C-terminus. Computer algorithms identified a putative helix-loop-helix motif that spanned the C0C1 cassettes of the C-terminus. These data suggest that the NR1 subunit may represent another member of a family of transmembrane proteins that undergo intramembrane proteolysis, releasing a cytosolic peptide that is actively translocated to the nucleus leading to alterations in gene regulation.     

Disruption of clh-1, a chloride channel gene, results in a wider body of Caenorhabditis elegans

We cloned the clh-1 gene coding for a putative ClC chloride channel in Caenorhabditis elegans. The gene product exhibited a high degree of homology with human ClC-1 and ClC-2. The clh-1 gene was predominantly expressed in the hypodermis, including seam cells. Null mutations of clh-1 caused a significantly wider body and an abnormal alae structure. High osmolarity in the culture medium restored the normal body width of the clh-1 mutants. These results suggest that the clh-1 gene contributes to maintenance of the body width through regulation of osmolarity.     

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.     

Mimicry of a host anion channel by a Helicobacter pylori pore-forming toxin

Bacterial pore-forming toxins have traditionally been thought to function either by causing an essentially unrestricted flux of ions and molecules across a membrane or by effecting the transmembrane transport of an enzymatically active bacterial peptide. However, the Helicobacter pylori pore-forming toxin, VacA, does not appear to function by either of these mechanisms, even though at least some of its effects in cells are dependent on its pore-forming ability. Here we show that the VacA channel exhibits two of the most characteristic electrophysiological properties of a specific family of cellular channels, the ClC channels: an open probability dependent on the molar ratio of permeable ions and single channel events resolvable as two independent, voltage-dependent transitions. The sharing of such peculiar properties by VacA and host ClC channels, together with their similar magnitudes of conductance, ion selectivities, and localization within eukaryotic cells, suggests a novel mechanism of toxin action in which the VacA pore largely mimics the electrophysiological behavior of a host channel, differing only in the membrane potential at which it closes. As a result, VacA can perturb, but not necessarily abolish, the homeostatic ionic imbalance across a membrane and so change cellular physiology without necessarily jeopardizing vitality.     

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.