Regulatory Phosphorylation Induces Extracellular Conformational Changes in a CLC Anion Channel

• Published in: Biophysical journal 2013-05, Vol.104 (9), p.1893-1904
• Main Authors: Yamada, Toshiki, Bhate, Manasi P., Strange, Kevin
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Cell cycle; Channels and Transporters; Chloride Channels - chemistry; Chloride Channels - genetics; Chloride Channels - metabolism; chlorides; Cytoplasm; HEK293 Cells; Humans; Ion Channel Gating; Molecular Dynamics Simulation; Molecular Sequence Data; mutants; Mutation; Phosphorylation; Protein Structure, Tertiary; Protein-Serine-Threonine Kinases - metabolism; Proteins; serine; Serine - genetics; Serine - metabolism; Signal transduction; transport proteins; zinc
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/j.bpj.2013.03.026

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 Zn2+ 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.