title: A comprehensive search for calcium binding sites critical for TMEM16A calcium-activated chloride channel activity.
abstract: TMEM16A forms calcium-activated chloride channels (CaCCs) that regulate physiological processes such as the secretions of airway epithelia and exocrine glands, the contraction of smooth muscles, and the excitability of neurons. Notwithstanding intense interest in the mechanism behind TMEM16A-CaCC calcium-dependent gating, comprehensive surveys to identify and characterize potential calcium sensors of this channel are still lacking. By aligning distantly related calcium-activated ion channels in the TMEM16 family and conducting systematic mutagenesis of all conserved acidic residues thought to be exposed to the cytoplasm, we identify four acidic amino acids as putative calcium-binding residues. Alterations of the charge, polarity, and size of amino acid side chains at these sites alter the ability of different divalent cations to activate the channel. Furthermore, TMEM16A mutant channels containing double cysteine substitutions at these residues are sensitive to the redox potential of the internal solution, providing evidence for their physical proximity and solvent accessibility.
authors: Department of Physiology, University of California, San Francisco, San Francisco, United States.
authors: Jason
authors: J
authors: Tien
authors: Department of Physiology, University of California, San Francisco, San Francisco, United States.
authors: Christian J
authors: CJ
authors: Peters
authors: Graduate Program in Chemistry and Chemical Biology, University of California, San Francisco, San Francisco, United States.
authors: Xiu Ming
authors: XM
authors: Wong
authors: Department of Physiology, University of California, San Francisco, San Francisco, United States.
authors: Tong
authors: T
authors: Cheng
authors: Department of Physiology, University of California, San Francisco, San Francisco, United States.
authors: Yuh Nung
authors: YN
authors: Jan
authors: Department of Physiology, University of California, San Francisco, San Francisco, United States.
authors: Lily Yeh
authors: LY
authors: Jan
authors: Department of Physiology, University of California, San Francisco, San Francisco, United States.
authors: Huanghe
authors: H
authors: Yang
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D000075369:Anoctamin-1; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D002147:Calmodulin / Q000737:chemistry; D018118:Chloride Channels / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D004330:Drosophila; D004594:Electrophysiology; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D007477:Ions; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D009363:Neoplasm Proteins / Q000737:chemistry*; D010084:Oxidation-Reduction; D017386:Sequence Homology, Amino Acid; D012997:Solvents / Q000737:chemistry; D013324:Strontium / Q000737:chemistry; D014982:Xenopus laevis
chemical_list: C476213:ANO1 protein, human; D000075369:Anoctamin-1; D002147:Calmodulin; D018118:Chloride Channels; D007477:Ions; D009363:Neoplasm Proteins; D012997:Solvents; D049452:Green Fluorescent Proteins; D003545:Cysteine; D000409:Alanine; D002118:Calcium; D013324:Strontium
keywords: ANO1 channels; TMEM16 channels; TMEM16A channels; biochemistry; biophysics; calcium activated chloride channels; calcium binding site; calcium dependent activation; frog; fruit fly; human; mouse; structural biology