title: Binding of ATP to the CBS domains in the C-terminal region of CLC-1.
abstract: The common gating of CLC-1 has been shown to be inhibited by intracellular adenosine triphosphate (ATP) in acidic pH conditions. Such modulation is thought to be mediated by direct binding of ATP to the cystathionine β-synthase (CBS) domains at the C-terminal cytoplasmic region of CLC-1. Guided by the crystal structure of the C-terminal domain of CLC-5, we constructed a homology model of CLC-1's C terminus and mutated critical amino acid residues lining the potential ATP-binding site. The CLC-1 mutations V634A and E865A completely abolished the ATP inhibition of CLC-1, consistent with the loss of ATP binding seen with the corresponding mutations in CLC-5. Mutating two other residues, V613 and V860, also disrupted the ATP modulation of CLC-1. However, placing aromatic amino acids at position 634 increases the apparent ATP affinity. Mutant cycle analyses showed that the modulation effects of ATP and cytidine triphosphate on wild-type CLC-1 and the V634F mutant were nonadditive, suggesting that the side chain of amino acid at position 634 interacts with the base moiety of the nucleotide. The mutation effects of V634F and V613A on the ATP modulation were also nonadditive, which is consistent with the assertion suggested from the homology model that these two residues may both interact with the bound nucleotide. These results provide evidence for a direct ATP binding for modulating the function of CLC-1 and suggest an overall conserved architecture of the ATP-binding sites in CLC-1 and CLC-5. This study also demonstrates that CLC-1 is a convenient experimental model for studying the interaction of nucleotides/nucleosides with the CBS domain.
authors: Center for Neuroscience, University of California, Davis, Davis, CA 95618, USA.
authors: Pang-Yen
authors: PY
authors: Tseng
authors: Wei-Ping
authors: WP
authors: Yu
authors: Hao-Yang
authors: HY
authors: Liu
authors: Xiao-Dong
authors: XD
authors: Zhang
authors: Xiaoqin
authors: X
authors: Zou
authors: Tsung-Yu
authors: TY
authors: Chen
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D018118:Chloride Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003541:Cystathionine beta-Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015640:Ion Channel Gating; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D014981:Xenopus / Q000378:metabolism
chemical_list: C086321:CLC-1 channel; D018118:Chloride Channels; D000255:Adenosine Triphosphate; D003541:Cystathionine beta-Synthase