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      A comprehensive search for calcium binding sites critical for TMEM16A calcium-activated chloride channel activity

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          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.

          DOI: http://dx.doi.org/10.7554/eLife.02772.001

          eLife digest

          Every cell in the body is surrounded by a barrier called the cell membrane. There are, however, a number of ways that molecules can pass through this membrane to either enter or leave the cell.

          Calcium-activated channels are a group of proteins that are embedded within the cell membrane and that allow different ions to pass through the membrane. These proteins are involved in a number of processes in a variety of tissues, for example in the gut, lungs and nervous system. A family of proteins called TMEM16 includes a number of calcium-activated channels that have been recently identified. However, it is not clear how these TMEM16 channel proteins detect the calcium ions that cause them to open.

          Two ideas have been suggested: the calcium ions might be detected by a protein called calmodulin, which then forces the channel to open; alternatively, the calcium ions might be detected by the channel protein itself. Tien, Peters et al. have now tested both of these ideas by focusing on a calcium-activated channel protein called TMEM16A, which allows chloride ions to pass through membranes.

          The possible role of calmodulin was tested in several ways, such as by preventing it from binding to the TMEM16A protein or from binding to calcium. However, none of these changes affected the opening of the channel; so Tien, Peters et al. concluded that calmodulin is not involved in these channels being activated by calcium ions.

          Next, Tien, Peters et al. tested specific parts of the TMEM16A channel protein itself to see if they were involved in calcium detection instead. Proteins are made from smaller building blocks called amino acids, and it is known that some amino acids are more likely to bind to calcium ions than others. There are 38 of these amino acids in the TMEM16A channel that are also found in other members of the TMEM16 family in both fruit flies and mammals. Tien, Peters et al. found that replacing five of these with other amino acids made the channel less sensitive to calcium. Further experiments suggested that four of these five amino acids are clustered at the site where a calcium ion might bind to the TMEM16A channel protein, which suggests that the protein itself can detect calcium directly.

          The next challenge will be to understand how calcium ions binding to the site on the TMEM16A channel protein can cause the channel to open to allow the chloride ions to pass through.

          DOI: http://dx.doi.org/10.7554/eLife.02772.002

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          Most cited references44

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          TMEM16A confers receptor-activated calcium-dependent chloride conductance.

          Calcium (Ca(2+))-activated chloride channels are fundamental mediators in numerous physiological processes including transepithelial secretion, cardiac and neuronal excitation, sensory transduction, smooth muscle contraction and fertilization. Despite their physiological importance, their molecular identity has remained largely unknown. Here we show that transmembrane protein 16A (TMEM16A, which we also call anoctamin 1 (ANO1)) is a bona fide Ca(2+)-activated chloride channel that is activated by intracellular Ca(2+) and Ca(2+)-mobilizing stimuli. With eight putative transmembrane domains and no apparent similarity to previously characterized channels, ANO1 defines a new family of ionic channels. The biophysical properties as well as the pharmacological profile of ANO1 are in full agreement with native Ca(2+)-activated chloride currents. ANO1 is expressed in various secretory epithelia, the retina and sensory neurons. Furthermore, knockdown of mouse Ano1 markedly reduced native Ca(2+)-activated chloride currents as well as saliva production in mice. We conclude that ANO1 is a candidate Ca(2+)-activated chloride channel that mediates receptor-activated chloride currents in diverse physiological processes.
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            TMEM16A, a membrane protein associated with calcium-dependent chloride channel activity.

            Calcium-dependent chloride channels are required for normal electrolyte and fluid secretion, olfactory perception, and neuronal and smooth muscle excitability. The molecular identity of these membrane proteins is still unclear. Treatment of bronchial epithelial cells with interleukin-4 (IL-4) causes increased calcium-dependent chloride channel activity, presumably by regulating expression of the corresponding genes. We performed a global gene expression analysis to identify membrane proteins that are regulated by IL-4. Transfection of epithelial cells with specific small interfering RNA against each of these proteins shows that TMEM16A, a member of a family of putative plasma membrane proteins with unknown function, is associated with calcium-dependent chloride current, as measured with halide-sensitive fluorescent proteins, short-circuit current, and patch-clamp techniques. Our results indicate that TMEM16A is an intrinsic constituent of the calcium-dependent chloride channel. Identification of a previously unknown family of membrane proteins associated with chloride channel function will improve our understanding of chloride transport physiopathology and allow for the development of pharmacological tools useful for basic research and drug development.
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              Expression cloning of TMEM16A as a calcium-activated chloride channel subunit.

              Calcium-activated chloride channels (CaCCs) are major regulators of sensory transduction, epithelial secretion, and smooth muscle contraction. Other crucial roles of CaCCs include action potential generation in Characean algae and prevention of polyspermia in frog egg membrane. None of the known molecular candidates share properties characteristic of most CaCCs in native cells. Using Axolotl oocytes as an expression system, we have identified TMEM16A as the Xenopus oocyte CaCC. The TMEM16 family of "transmembrane proteins with unknown function" is conserved among eukaryotes, with family members linked to tracheomalacia (mouse TMEM16A), gnathodiaphyseal dysplasia (human TMEM16E), aberrant X segregation (a Drosophila TMEM16 family member), and increased sodium tolerance (yeast TMEM16). Moreover, mouse TMEM16A and TMEM16B yield CaCCs in Axolotl oocytes and mammalian HEK293 cells and recapitulate the broad CaCC expression. The identification of this new family of ion channels may help the development of CaCC modulators for treating diseases including hypertension and cystic fibrosis.
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                Author and article information

                Contributors
                Role: Reviewing editor
                Journal
                eLife
                Elife (Cambridge)
                eLife
                eLife
                eLife
                eLife Sciences Publications, Ltd
                2050-084X
                30 June 2014
                2014
                : 3
                : e02772
                Affiliations
                [1 ]Department of Physiology, University of California, San Francisco , San Francisco, United States
                [2 ]Graduate Program in Chemistry and Chemical Biology, University of California, San Francisco , San Francisco, United States
                [3 ]Department of Biochemistry and Biophysics, University of California, San Francisco , San Francisco, United States
                [4 ]Howard Hughes Medical Institute, University of California, San Fransisco , San Fransisco, United States
                The University of Texas at Austin , United States
                The University of Texas at Austin , United States
                Author notes
                [* ]For correspondence: Lily.Jan@ 123456ucsf.edu (LYJ);
                [* ]For correspondence: huanghe.yang@ 123456ucsf.edu (HY)
                [†]

                These authors contributed equally to this work.

                [‡]

                Institute of Molecular and Cell Biology, Singapore, Singapore.

                Article
                02772
                10.7554/eLife.02772
                4112547
                24980701
                cb1897ed-e99b-47cf-90e3-624ce9ff06ea
                Copyright © 2014, Tien et al

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

                History
                : 12 March 2014
                : 28 June 2014
                Funding
                Funded by: National Institutes of Health FundRef identification ID: http://dx.doi.org/10.13039/100000002
                Award ID: 5F31NS076180
                Award Recipient :
                Funded by: Howard Hughes Medical Institute FundRef identification ID: http://dx.doi.org/10.13039/100000011
                Award Recipient :
                Funded by: Heart and Stroke Foundation of Canada FundRef identification ID: http://dx.doi.org/10.13039/100004411
                Award Recipient :
                Funded by: National Institutes of Health FundRef identification ID: http://dx.doi.org/10.13039/100000002
                Award ID: R01NS069229
                Award Recipient :
                Funded by: National Institutes of Health FundRef identification ID: http://dx.doi.org/10.13039/100000002
                Award ID: K99NS086916
                Award Recipient :
                The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
                Categories
                Research Article
                Biochemistry
                Biophysics and Structural Biology
                Custom metadata
                0.7
                The identification of four acidic amino acids as potential calcium-binding residues in the TMEM16A calcium-activated chloride channel furthers the molecular understanding of this ion channel family.

                Life sciences
                tmem16 channels,calcium activated chloride channels,calcium binding site,tmem16a channels,ano1 channels,calcium dependent activation,human,mouse,frog,fruit fly

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