Oleic acid blocks the calcium-activated chloride channel TMEM16A/ANO1.

The calcium-activated chloride channel TMEM16A (ANO1) supports the passive movement of chloride ions across membranes and controls critical cell functions. Here we study the block of wild-type and mutant TMEM16A channels expressed in HEK293 cells by oleic acid, a monounsaturated omega-9 fatty acid beneficial for cardiovascular health. We found that oleic acid irreversibly blocks TMEM16A in a dose- and voltage-dependent manner at low intracellular Ca2+. We tested whether oleic acid interacted with the TMEM16A pore, varying the permeant anion concentration and mutating pore residues. Lowering the permeating anion concentration in the intracellular side did nothing but the blockade was intensified by increasing the anion concentration in the extracellular side. However, the blockade of the pore mutants E633A and I641A was voltage-independent, and the I641A IC50, a mutant with the inner hydrophobic gate in disarray, increased 16-fold. Furthermore, the uncharged methyl-oleate blocked 20-24% of the wild-type and I641A channels regardless of voltage. Our findings suggest that oleic acid inhibits TMEM16A by an allosteric mechanism after the electric field drives oleic acid's charged moiety inside the pore. Block of TMEM16A might be why oleic acid has a beneficial impact on the cardiovascular system.

Phosphatidylinositol 4,5-bisphosphate, cholesterol, and fatty acids modulate the calcium-activated chloride channel TMEM16A (ANO1).

The TMEM16A-mediated Ca2+-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+. 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-ß-cyclodextrin M-ßCD) or restoration (with M-ßCD+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-ßCD alone transiently increases TMEM16A activity and dampens rundown whereas M-ßCD+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-ßCD, M-ßCD+cholesterol, oleic, or docosahexaenoic acids. Thus, we propose that lipids and fatty acids regulate TMEM16A channels through a membrane-delimited protein-lipid interaction.