Regulatory Conformational Coupling between CLC Anion Channel Membrane and Cytoplasmic Domains.
CLC anion channels are homodimeric proteins. Each subunit is comprised of 18 α-helices designated "A-R" and an intracellular carboxy-terminus containing two cystathionine-β-synthase (CBS1 and CBS2) domains. Conformational coupling between membrane and intracellular domains via poorly understood mechanisms is required for CLC regulation. The activity of the C. elegans CLC channel CLH-3b is reduced by phosphorylation of a carboxy-terminus "activation domain," which disrupts its interaction with CBS domains. CBS2 interfaces with a short intracellular loop, the H-I loop, connecting membrane helices H and I. Alanine mutation of a conserved H-I loop tyrosine residue, Y232, prevents regulation demonstrating that the loop functions to couple phosphorylation-dependent CBS domain conformational changes to channel membrane domains. To gain further insight into the mechanisms of this coupling, we mutated conserved amino acid residues in membrane helices H and I. Only mutation of the H-helix valine residue V228 to leucine prevented phosphorylation-dependent channel regulation. Structural and functional studies of other CLC proteins suggest that V228 may interact with Y529, a conserved R-helix tyrosine residue that forms part of the CLC ion conduction pathway. Mutation of Y529 to alanine also prevented CLH-3b regulation. Intracellular application of the sulfhydryl reactive reagent MTSET using CLH-3b channels engineered with single-cysteine residues in CBS2 indicate that V228L, Y529A, and Y232A disrupt putative regulatory intracellular conformational changes. Extracellular Zn2+inhibits CLH-3b and alters the effects of intracellular MTSET on channel activity. The effects of Zn2+are disrupted by V228L, Y529A, and Y232A. Collectively, our findings indicate that there is conformational coupling between CBS domains and the H and R membrane helices mediated by the H-I loop. We propose a simple model by which conformational changes in H and R helices mediate CLH-3b regulation by activation domain phosphorylation.
MDI Biological Laboratory, Kathryn W. Davis Center for Regenerative Biology and Medicine, Salisbury Cove, Maine.
Toshiki
T
Yamada
MDI Biological Laboratory, Kathryn W. Davis Center for Regenerative Biology and Medicine, Salisbury Cove, Maine. Electronic address: kstrange@mdibl.org.
Kevin
K
Strange
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D018118:Chloride Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D003593:Cytoplasm / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D000072417:Protein Domains; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism
D018118:Chloride Channels; D021122:Protein Subunits