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      Structural basis for anion conduction in the calcium-activated chloride channel TMEM16A


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          The calcium-activated chloride channel TMEM16A is a member of a conserved protein family that comprises ion channels and lipid scramblases. Although the structure of the scramblase nhTMEM16 has defined the architecture of the family, it was unknown how a channel has adapted to cope with its distinct functional properties. Here we have addressed this question by the structure determination of mouse TMEM16A by cryo-electron microscopy and a complementary functional characterization. The protein shows a similar organization to nhTMEM16, except for changes at the site of catalysis. There, the conformation of transmembrane helices constituting a membrane-spanning furrow that provides a path for lipids in scramblases has changed to form an enclosed aqueous pore that is largely shielded from the membrane. Our study thus reveals the structural basis of anion conduction in a TMEM16 channel and it defines the foundation for the diverse functional behavior in the TMEM16 family.

          DOI: http://dx.doi.org/10.7554/eLife.26232.001

          eLife digest

          Cell membranes are made up of two layers of oily molecules, called lipids, embedded with a variety of proteins. Each type of membrane protein carries out a particular activity for the cell, and many are involved in transporting other molecules from one side of the membrane to the other.

          The TMEM16 proteins are a large family of membrane proteins. Most are known as lipid scramblases and move lipids between the two layers of the membrane. However, some TMEM16 proteins transport ions in or out of the cell, and are instead called ion channels. TMEM16 proteins are found in animals, plants and fungi but not bacteria, and play key roles in many biological activities that keep these organisms alive. For example, in humans, ion channels belonging to the TMEM16 family help keep the lining of the lung moist, and allow muscles in the gut to contract.

          The structure of a scramblase shows that two protein units interact, with each unit containing a furrow that spans the membrane, through which lipids can move from one layer to the other. However, to date, the shape of a TMEM16 ion channel has not been determined. It was therefore not clear how a protein with features that let it transport large, oily molecules like lipids had evolved to transport small, charged particles instead.

          TMEM16A is a member of the TMEM16 family that transports negatively charged chloride ions. Using a technique called cryo-electron microscopy, Paulino et al. have determined the three-dimensional shape of the version of TMEM16A from a mouse. Overall, TMEM16A is organized similarly to the lipid scramblase. However, some parts of the TMEM16A protein have undergone rearrangements such that the membrane-exposed furrow that provides a path for lipids in scramblases is now partially sealed in TMEM16A. This results in an enclosed pore that is largely shielded from the oily membrane and through which ions can pass. Additionally, biochemical analysis suggests that TMEM16A forms a narrow pore that may widen towards the side facing the inside of the cell, though further work is needed to understand if this is relevant to the protein’s activity.

          The three-dimensional structure of TMEM16A reveals how the protein’s architecture differs from other family members working as lipid scramblases. It also gives insight into how TMEM16 proteins might work as ion channels. These findings can now form a strong basis for future studies into the activity of TMEM16 proteins.

          DOI: http://dx.doi.org/10.7554/eLife.26232.002

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          Most cited references36

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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.

                Author and article information

                Role: Reviewing editor
                eLife Sciences Publications, Ltd
                31 May 2017
                : 6
                : e26232
                [1 ]deptDepartment of Biochemistry , University of Zurich , Zurich, Switzerland
                University of Wisconsin-Madison , United States
                University of Wisconsin-Madison , United States
                Author notes

                Department Biozentrum, University of Basel, Basel, Switzerland.


                Laboratory of Biomolecular Research, Paul Scherrer Institute, Villigen, Switzerland.

                Author information
                © 2017, Paulino 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.

                : 21 February 2017
                : 11 April 2017
                Funded by: FundRef http://dx.doi.org/10.13039/100011102, Seventh Framework Programme;
                Award ID: 339116 AnoBest
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100006447, Universität Zürich;
                Award ID: Forschungskredit FK-16-036
                Award Recipient :
                The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
                Research Article
                Biophysics and Structural Biology
                Custom metadata
                Single-particle cryo-EM and electrophysiology studies of the chloride channel TMEM16A reveals the structural basis for anion conduction and uncover its relationship to lipid scramblases of the same family.

                Life sciences
                ligand gated ion channels,cryo-electron microscopy,patch-clamp electrophsiology,ion permeation,mouse


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