The TMEM16A-mediated Ca-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 Ca. 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+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+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+cholesterol, oleic, or docosahexaenoic acids. Thus, we propose that lipids and fatty acids regulate TMEM16A channels through a membrane-delimited protein-lipid interaction.

Intracellular permeant anions, and not extracellular protons, are the predominant driver of fast gating in the hyperpolarization-activated CLC-2 chloride channel.

Key pointsr Calcium-activated chloride channels TMEM16A and TMEM16B support important physiological processes such as fast block of polyspermy, fluid secretion, control of blood pressure and sensory transduction.r Given the physiological importance of TMEM16 channels, it is important to study how incoming stimuli activate these channels. Here we study how channels open and close and how the process of gating is regulated.r We show that TMEM16A and TMEM16B display fast and slow gating. These gating modes are regulated by voltage and external chloride.r Dual gating explains the complex time course of the anion current. r Residues within the first intracellular loop of the channel influence the slow gating mode. r Dual gating is an intrinsic property observed in endogenous calcium-activated chloride channels and could be relevant to physiological processes that require sustained chloride ion movement.Abstract TMEM16A and TMEM16B are molecular components of the physiologically relevant calcium-activated chloride channels (CaCCs) present in many tissues. Their gating is dictated by membrane voltage (V m ), intracellular calcium concentrations ([Ca 2+ ] i ) and external permeant anions. As a consequence, the chloride current (I Cl ) kinetics is complex. For example, TMEM16A I Cl activates slowly with a non-mono-exponential time course while TMEM16B I Cl activates rapidly following a mono-exponential behaviour. To understand the underlying mechanism responsible for the complex activation kinetics, we recorded I Cl from HEK-293 cells transiently transfected with either TMEM16A or TMEM16B as well as from mouse parotid acinar cells. Two distinct V m -dependent gating modes were uncovered: a fast-mode on the millisecond time scale followed by a slow mode on the second time scale. Using long (20 s) depolarizing pulses both gating modes were activated, and a slowly rising I Cl was recorded in whole-cell and inside-out patches. The amplitude of I Cl at the end of the long pulse nearly doubled and was blocked by 100 μM tannic acid. The slow gating mode was strongly reduced by decreasing the [Cl − ] o from 140 to 30 mM and by altering the sequence of the first intracellular loop. Mutating 480 RSQ 482 to AVK in the first intracellular loop of TMEM16B nearly abolished slow gating, but, mutating 448 AVK 451 to RSQ in TMEM16A has little effect. Deleting 448 EAVK 451 residues in TMEM16A reduced slow gating. We conclude that TMEM16 CaCCs have intrinsic V m -and Cl − -sensitive dual gating that elicits complex I Cl kinetics.

TMEM16A (ANO1), the pore-forming subunit of calcium-activated chloride channels, regulates several physiological and pathophysiological processes such as smooth muscle contraction, cardiac and neuronal excitability, salivary secretion, tumour growth, and cancer progression. Gating of TMEM16A is complex because it involves the interplay between increases in intracellular calcium concentration ([Ca2+]i), membrane depolarization, extracellular Cl− or permeant anions, and intracellular protons. Our goal here was to understand how these variables regulate TMEM16A gating and to explain four observations. a) TMEM16A is activated by voltage in the absence of intracellular Ca2+. b) The Cl− conductance is decreased after reducing extracellular Cl− concentration ([Cl−]o). c) ICl is regulated by physiological concentrations of [Cl−]o. d) In cells dialyzed with 0.2 µM [Ca2+]i, Cl− has a bimodal effect: at [Cl−]o < 30 mM TMEM16A current activates with a monoexponential time course, but above 30 mM [Cl−]o ICl activation displays fast and slow kinetics. To explain the contribution of Vm, Ca2+ and Cl− to gating, we developed a 12-state Markov chain model. This model explains TMEM16A activation as a sequential, direct, and Vm-dependent binding of two Ca2+ ions coupled to a Vm-dependent binding of an external Cl− ion, with Vm-dependent transitions between states. Our model predicts that extracellular Cl− does not alter the apparent Ca2+ affinity of TMEM16A, which we corroborated experimentally. Rather, extracellular Cl− acts by stabilizing the open configuration induced by Ca2+ and by contributing to the Vm dependence of activation.

Transmembrane protein 16A (TMEM16A), also known as ANO1, the pore-forming subunit of a Ca -dependent Cl channel (CaCC), is activated by direct, voltage-dependent, binding of intracellular Ca . Endogenous CaCCs are regulated by extracellular protons; however, the molecular basis of such regulation remains unidentified. Here, we evaluated the effects of different extracellular proton concentrations ([H ] ) on mouse TMEM16A expressed in HEK-293 cells using whole-cell and inside-out patch-clamp recordings. We found that increasing the [H ] from 10 to 10 m caused a progressive increase in the chloride current (I ) that is described by titration of a protonatable site with pK = 7.3. Protons regulate TMEM16A in a voltage-independent manner, regardless of channel state (open or closed), and without altering its apparent Ca sensitivity. Noise analysis showed that protons regulate TMEM16A by tuning its open probability without modifying the single channel current. We found a robust reduction of the proton effect at high [Ca ] . To identify protonation targets we mutated all extracellular glutamate and histidine residues and 4 of 11 aspartates. Most mutants were sensitive to protons. However, mutation that substituted glutamic acid (E) for glutamine (Q) at amino acid position 623 (E623Q) displayed a titration curve shifted to the left relative to wild type channels and the I was nearly insensitive to proton concentrations between 10 and 10 m. Additionally, I of the mutant containing an aspartic acid (D) to asparagine (N) substitution at position 405 (D405N) mutant was partially inhibited by a proton concentration of 10 m, but 10 m produced the same effect as in wild type. Based on our findings we propose that external protons titrate glutamic acid 623, which enables voltage activation of TMEM16A at non-saturating [Ca ] .