1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics
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A comprehensive search for calcium binding sites critical for TMEM16A calcium-activated chloride channel activity

  1. Jason Tien
  2. Christian J Peters
  3. Xiu Ming Wong
  4. Tong Cheng
  5. Yuh Nung Jan
  6. Lily Yeh Jan
  7. Huanghe Yang  Is a corresponding author
  1. University of California, San Francisco, United States
Research Article
  • Cited 64
  • Views 2,797
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Cite this article as: eLife 2014;3:e02772 doi: 10.7554/eLife.02772

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.

Article and author information

Author details

  1. Jason Tien

    University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Christian J Peters

    University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Xiu Ming Wong

    University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Tong Cheng

    University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Yuh Nung Jan

    University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Lily Yeh Jan

    University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Huanghe Yang

    University of California, San Francisco, San Francisco, United States
    For correspondence
    huanghe.yang@ucsf.edu
    Competing interests
    The authors declare that no competing interests exist.

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the female Xenopus laevis were handled according to approved institutional animal care and use committee (IACUC) protocol (#AN086415-03A) of the University of California, San Francisco. The procedures for harvesting oocytes and housing animals were performed in strict accordance with the protocol, and every effort was made to minimize suffering.

Reviewing Editor

  1. Richard Aldrich, The University of Texas at Austin, United States

Publication history

  1. Received: March 12, 2014
  2. Accepted: June 28, 2014
  3. Accepted Manuscript published: June 30, 2014 (version 1)
  4. Version of Record published: July 29, 2014 (version 2)

Copyright

© 2014, Tien et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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