title: Unique gating properties of C. elegans ClC anion channel splice variants are determined by altered CBS domain conformation and the R-helix linker.
abstract: 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.
authors.affiliation: Boylan Center for Cellular and Molecular Physiology, Mount Desert Island Biological Laboratory, Salisbury Cove, ME, USA.
authors.forename: Sonya
authors.initials: S
authors.lastname: Dave
authors.forename: Jonathan H
authors.initials: JH
authors.lastname: Sheehan
authors.forename: Jens
authors.initials: J
authors.lastname: Meiler
authors.forename: Kevin
authors.initials: K
authors.lastname: Strange
mesh_terms: D000595:Amino Acid Sequence; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D018118:Chloride Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D003593:Cytoplasm / Q000378:metabolism*; D006801:Humans; D015640:Ion Channel Gating; D007700:Kinetics; D008564:Membrane Potentials; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D017354:Point Mutation; D011487:Protein Conformation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D014162:Transfection
chemical_list: D029742:Caenorhabditis elegans Proteins; D018118:Chloride Channels; D020033:Protein Isoforms; C417201:clh-3 protein, C elegans