CLC anion channel regulatory phosphorylation and conserved signal transduction domains.
The signaling mechanisms that regulate CLC anion channels are poorly understood. Caenorhabditis elegans CLH-3b is a member of the CLC-1/2/Ka/Kb channel subfamily. CLH-3b is activated by meiotic cell-cycle progression and cell swelling. Inhibition is brought about by GCK-3 kinase-mediated phosphorylation of S742 and S747 located on a ∼176 amino acid disordered domain linking CBS1 and CBS2. Much of the inter-CBS linker is dispensable for channel regulation. However, deletion of a 14 amino acid activation domain encompassing S742 and S747 inhibits channel activity to the same extent as GCK-3. The crystal structure of CmCLC demonstrated that CBS2 interfaces extensively with an intracellular loop connecting membrane helices H and I, the C-terminus of helix D, and a short linker connecting helix R to CBS1. Point mutagenesis of this interface identified two highly conserved aromatic amino acid residues located in the H-I loop and the first α-helix (α1) of CBS2. Mutation of either residue to alanine rendered CLH-3b insensitive to GCK-3 inhibition. We suggest that the dephosphorylated activation domain normally interacts with CBS1 and/or CBS2, and that conformational information associated with this interaction is transduced through a conserved signal transduction module comprising the H-I loop and CBS2 α1.
Boylan Center for Cellular and Molecular Physiology, Mount Desert Island Biological Laboratory, Salisbury Cove, Maine, USA.
Hiroaki
H
Miyazaki
Toshiki
T
Yamada
Angela
A
Parton
Rebecca
R
Morrison
Sunghoon
S
Kim
Albert H
AH
Beth
Kevin
K
Strange
D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D020134:Catalytic Domain; D018118:Chloride Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D003541:Cystathionine beta-Synthase / Q000737:chemistry / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D054730:Protein Interaction Domains and Motifs; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D015398:Signal Transduction
D029742:Caenorhabditis elegans Proteins; D018118:Chloride Channels; C417201:clh-3 protein, C elegans; D017346:Protein Serine-Threonine Kinases; C527979:gck-3 protein, C elegans; D003541:Cystathionine beta-Synthase