A comprehensive search for calcium binding sites critical for TMEM16A calcium-activated chloride channel activity
Jason Tien,
Christian J Peters,
Xiu Ming Wong,
Tong Cheng,
Yuh Nung Jan,
Lily Yeh Jan,
Huanghe Yang
Affiliations
Jason Tien
Department of Physiology, University of California, San Francisco, San Francisco, United States
Christian J Peters
Department of Physiology, University of California, San Francisco, San Francisco, United States
Xiu Ming Wong
Graduate Program in Chemistry and Chemical Biology, University of California, San Francisco, San Francisco, United States
Tong Cheng
Department of Physiology, University of California, San Francisco, San Francisco, United States; Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States; Howard Hughes Medical Institute, University of California, San Fransisco, San Fransisco, United States
Yuh Nung Jan
Department of Physiology, University of California, San Francisco, San Francisco, United States; Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States; Howard Hughes Medical Institute, University of California, San Fransisco, San Fransisco, United States
Lily Yeh Jan
Department of Physiology, University of California, San Francisco, San Francisco, United States; Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States; Howard Hughes Medical Institute, University of California, San Fransisco, San Fransisco, United States
Huanghe Yang
Department of Physiology, University of California, San Francisco, San Francisco, United States; Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States; Howard Hughes Medical Institute, University of California, San Fransisco, San Fransisco, United States
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.
