title: Regulatory phosphorylation induces extracellular conformational changes in a CLC anion channel.
abstract: CLH-3b is a CLC-1/2/Ka/Kb channel homolog activated by meiotic cell cycle progression and cell swelling. Channel inhibition occurs by GCK-3 kinase-mediated phosphorylation of serine residues on the cytoplasmic C-terminus linker connecting CBS1 and CBS2. Two conserved aromatic amino acid residues located on the intracellular loop connecting membrane helices H and I and α1 of CBS2 are required for transducing phosphorylation changes into changes in channel activity. Helices H and I form part of the interface between the two subunits that comprise functional CLC channels. Using a cysteine-less CLH-3b mutant, we demonstrate that the sulfhydryl reagent reactivity of substituted cysteines at the subunit interface changes dramatically during GCK-3-mediated channel inhibition and that these changes are prevented by mutation of the H-I loop/CBS2 α1 signal transduction domain. We also show that GCK-3 modifies Zn(2+) inhibition, which is thought to be mediated by the common gating process. These and other results suggest that phosphorylation of the cytoplasmic C-terminus inhibits CLH-3b by inducing subunit interface conformation changes that activate the common gate. Our findings have important implications for understanding CLC regulation by diverse signaling mechanisms and for understanding the structure/function relationships that mediate intraprotein communication in this important family of Cl(-) transport proteins.
authors.affiliation: Boylan Center for Cellular and Molecular Physiology, Mount Desert Island Biological Laboratory, Salisbury Cove, Maine, USA.
authors.forename: Toshiki
authors.initials: T
authors.lastname: Yamada
authors.forename: Manasi P
authors.initials: MP
authors.lastname: Bhate
authors.forename: Kevin
authors.initials: K
authors.lastname: Strange
mesh_terms: D000595:Amino Acid Sequence; D018118:Chloride Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012694:Serine / Q000235:genetics / Q000378:metabolism
chemical_list: D018118:Chloride Channels; C115376:ClC-3 channel; D012694:Serine; D017346:Protein Serine-Threonine Kinases