Phosphatidylinositol 4,5-bisphosphate, cholesterol, and fatty acids modulate the calcium-activated chloride channel TMEM16A (ANO1)
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The TMEM16A-mediated Ca2%2b-activated Cl− current drives several important physiological functions. Membrane lipids regulate ion channels and transporters but their influence on members of the TMEM16 family is poorly understood. Here we have studied the regulation of TMEM16A by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), cholesterol, and fatty acids using patch clamp, biochemistry and fluorescence microscopy. We found that depletion of membrane PI(4,5)P2 causes a decline in TMEM16A current that is independent of cytoskeleton, but is partially prevented by removing intracellular Ca2%2b. On the other hand, supplying PI(4,5)P2 to inside-out patches attenuated channel rundown and/or partially rescued activity after channel rundown. Also, depletion (with methyl-β-cyclodextrin M-βCD) or restoration (with M-βCD %2b cholesterol) of membrane cholesterol slows down the current decay observed after reduction of PI(4,5)P2. Neither depletion nor restoration of cholesterol change PI(4,5)P2 content. However, M-βCD alone transiently increases TMEM16A activity and dampens rundown whereas M-βCD %2b cholesterol increases channel rundown. Thus, PI(4,5)P2 is required for TMEM16A function while cholesterol directly and indirectly via a PI(4,5)P2-independent mechanism regulate channel function. Stearic, arachidonic, oleic, docosahexaenoic, and eicosapentaenoic fatty acids as well as methyl stearate inhibit TMEM16A in a dose- and voltage-dependent manner. Phosphatidylserine, a phospholipid whose hydrocarbon tails contain stearic and oleic acids also inhibits TMEM16A. Finally, we show that TMEM16A remains in the plasma membrane after treatment with M-βCD, M-βCD %2b cholesterol, oleic, or docosahexaenoic acids. Thus, we propose that lipids and fatty acids regulate TMEM16A channels through a membrane-delimited protein-lipid interaction. © 2017 Elsevier B.V.
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Consejo Nacional de Ciencia y Tecnología, CONACYT: 219949, 220224, 234820, 256034, 290807, FDC 2016-01-1955, IN206616 Grant
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National Eye Institute, NEI: R01EY014852 Grant
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National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIAMS: R01AR067786 Grant
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National Institutes of Health, NIH: R01-EY0114852 Grant
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keywords
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Anoctamin; Fluorescence microscopy; Lipids; Patch clamp; Poly-unsaturated fatty acids; Regulation; TMEM16A arachidonic acid; calcium; calcium activated chloride channel; cholesterol; docosahexaenoic acid; fatty acid; icosapentaenoic acid; methyl beta cyclodextrin; oleic acid; phosphatidylinositol 4,5 bisphosphate; phosphatidylserine; stearic acid; TMEM16A protein; unclassified drug; ANO1 protein, human; anoctamin 1; calcium; cholesterol; fatty acid; phosphatidylinositol 4,5 bisphosphate; tumor protein; Article; biochemistry; calcium cell level; cell membrane; chloride current; controlled study; cytoskeleton; dose response; down regulation; fluorescence microscopy; inside out patch clamp; lipid membrane; priority journal; protein function; protein lipid interaction; regulatory mechanism; calcium signaling; genetics; HEK293 cell line; human; metabolism; physiology; Anoctamin-1; Calcium; Calcium Signaling; Cell Membrane; Cholesterol; Fatty Acids; HEK293 Cells; Humans; Neoplasm Proteins; Phosphatidylinositol 4,5-Diphosphate
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