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      Cryo-EM structures and functional characterization of the murine lipid scramblase TMEM16F

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          Abstract

          The lipid scramblase TMEM16F initiates blood coagulation by catalyzing the exposure of phosphatidylserine in platelets. The protein is part of a family of membrane proteins, which encompasses calcium-activated channels for ions and lipids. Here, we reveal features of murine TMEM16F (mTMEM16F) that underlie its function as a lipid scramblase and an ion channel. The cryo-EM data of mTMEM16F in absence and presence of Ca 2+ define the ligand-free closed conformation of the protein and the structure of a Ca 2+-bound intermediate. Both conformations resemble their counterparts of the scrambling-incompetent anion channel mTMEM16A, yet with distinct differences in the region of ion and lipid permeation. In conjunction with functional data, we demonstrate the relationship between ion conduction and lipid scrambling. Although activated by a common mechanism, both functions appear to be mediated by alternate protein conformations that are at equilibrium in the ligand-bound state.

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          Most cited references47

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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
                Role: Senior Editor
                Journal
                eLife
                Elife
                eLife
                eLife
                eLife Sciences Publications, Ltd
                2050-084X
                20 February 2019
                2019
                : 8
                : e44365
                Affiliations
                [1 ]deptDepartment of Biochemistry University of Zurich ZurichSwitzerland
                [2 ]deptDepartment of Structural Biology at the Groningen Biomolecular Sciences and Biotechnology Institute University of Groningen GroningenNetherlands
                National Institute of Neurological Disorders and Stroke, National Institutes of Health United States
                The University of Texas at Austin United States
                National Institute of Neurological Disorders and Stroke, National Institutes of Health United States
                United States
                National Institute of Neurological Disorders and Stroke, National Institutes of Health United States
                Memorial Sloan Kettering Cancer Center United States
                Author notes
                [†]

                These authors contributed equally to this work.

                Author information
                http://orcid.org/0000-0001-8446-1098
                http://orcid.org/0000-0001-5098-929X
                https://orcid.org/0000-0001-8013-0144
                http://orcid.org/0000-0001-6816-136X
                http://orcid.org/0000-0002-2193-6129
                http://orcid.org/0000-0001-7017-109X
                Article
                44365
                10.7554/eLife.44365
                6414204
                30785399
                23fffafe-0f7a-4c83-bc27-a61b9d247e40
                © 2019, Alvadia 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 December 2018
                : 19 February 2019
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100003246, Nederlandse Organisatie voor Wetenschappelijk Onderzoek;
                Award ID: 740.018.016
                Award Recipient :
                Funded by: FP7 European Research Council;
                Award ID: 339116, AnoBest
                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
                Structural Biology and Molecular Biophysics
                Custom metadata
                While activated by a common mechanism, both functions in TMEM16F - lipid scrambling and ion conduction - are likely mediated by alternate protein conformations that are at equilibrium in the ligand-bound state.

                Life sciences
                cryo-em,tmem16f,lipid scramblase,ion conduction,electrophysiology,mechanism of action,human,mouse

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