title: Carboxy terminus splice variation alters ClC channel gating and extracellular cysteine reactivity.
abstract: CLH-3a and CLH-3b are Caenorhabditis elegans ClC channel splice variants that exhibit striking differences in voltage, Cl(-), and H(+) sensitivity. The major primary structure differences between the channels include a 71 amino acid CLH-3a N-terminal extension and a 270 amino acid extension of the CLH-3b C-terminus. Deletion of the CLH-3a N-terminus or generation of a CLH-3a/b chimera has no effect on channel gating. In contrast, deletion of a 169 amino acid C-terminal CLH-3b splice insert or deletion of the last 11 amino acids of cystathionine-beta-synthase domain 1 gives rise to functional properties identical to those of CLH-3a. Voltage-, Cl(-)-, and H(+)-dependent gating of both channels are lost when their glutamate gates are mutated to alanine. Glutamate gate cysteine mutants exhibit similar degrees of inhibition by MTSET, but the inhibition time constant of CLH-3b is sevenfold greater than that of CLH-3a. Differences in MTSET inhibition are reversed by deletion of the same cytoplasmic C-terminal regions that alter CLH-3b gating. Our results indicate that splice variation of the CLH-3b cytoplasmic C-terminus alters extracellular structure and suggest that differences in the conformation of the outer pore vestibule and associated glutamate gate may account for differences in CLH-3a and CLH-3b gating.
authors.affiliation: Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2520, USA.
authors.forename: Liping
authors.initials: L
authors.lastname: He
authors.forename: Jerod
authors.initials: J
authors.lastname: Denton
authors.forename: Keith
authors.initials: K
authors.lastname: Nehrke
authors.forename: Kevin
authors.initials: K
authors.lastname: Strange
mesh_terms: D019943:Amino Acid Substitution; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D018118:Chloride Channels / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004274:DNA, Recombinant / Q000235:genetics; D014644:Genetic Variation; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D007668:Kidney / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D013329:Structure-Activity Relationship
chemical_list: D029742:Caenorhabditis elegans Proteins; D018118:Chloride Channels; D004274:DNA, Recombinant; C417201:clh-3 protein, C elegans; D003545:Cysteine