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TMEM16A (ANO1), the pore forming subunit of a Ca2+-dependent Cl− channel (CaCC), is activated by direct, voltage-dependent, binding of intracellular Ca2+. 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+]o) on mouse TMEM16A expressed in HEK-293 cells using whole-cell and inside-out patch clamp recordings. We found that increasing the [H+]o from 10−10 to 10−5.5 M caused a progressive increase in the chloride current (ICl) 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 Ca2+ 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 [Ca2+]i. To identify protonation targets we mutated all extracellular glutamate and histidine residues and four of eleven aspartates. Most mutants were sensitive to protons. However, E623Q displayed a titration curve shifted to the left relative to WT channels and the ICl was nearly insensitive to proton concentrations between 10−5.5 and 10−9.0 M. Additionally, ICl of the D405N mutant was partially inhibited by a proton concentration of 10−5.5 M, but 10−9.0 M produced the same effect as in WT. Based on our findings we propose that external protons titrate glutamic acid 623, which enables voltage activation of TMEM16A at non-saturating [Ca2+]i. This article is protected by copyright. All rights reserved

Key points 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. 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. We show that TMEM16A and TMEM16B display fast and slow gating. These gating modes are regulated by voltage and external chloride. Dual gating explains the complex time course of the anion current. Residues within the first intracellular loop of the channel influence the slow gating mode. 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 (Vm), intracellular calcium concentrations ([Ca2+]i) and external permeant anions. As a consequence, the chloride current (ICl) kinetics is complex. For example, TMEM16A ICl activates slowly with a non-mono-exponential time course while TMEM16B ICl activates rapidly following a mono-exponential behaviour. To understand the underlying mechanism responsible for the complex activation kinetics, we recorded ICl from HEK-293 cells transiently transfected with either TMEM16A or TMEM16B as well as from mouse parotid acinar cells. Two distinct Vm-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 ICl was recorded in whole-cell and inside-out patches. The amplitude of ICl 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 480RSQ482 to AVK in the first intracellular loop of TMEM16B nearly abolished slow gating, but, mutating 448AVK451 to RSQ in TMEM16A has little effect. Deleting 448EAVK451 residues in TMEM16A reduced slow gating. We conclude that TMEM16 CaCCs have intrinsic Vm- and Cl−-sensitive dual gating that elicits complex ICl kinetics.

Mouse parotid acinar cells express P2X4 and P2X7 receptors (mP2X4R and mP2X7R) whose physiological function remains undetermined. Here we show that mP2X4R expressed in HEK-293 cells do not allow the passage of tetraethylammonium (TEA+) and promote little, if any, ethidium bromide (EtBr) uptake when stimulated with ATP or BzATP. In contrast, mP2X7R generates slowly decaying TEA+ current, sustained Na+ current and promotes robust EtBr uptake. However, ATP-activated TEA+ current from acinar cells was unlike that generated by mP2X7R or mP2X4R. Functional interactions between mP2X4R and mP2X7R were investigated in HEK cells co-transfected with different mP2X4 : mP2X7 cDNA ratios and using solutions containing either TEA+ or Na+ ions. Co-expressed channels generated a TEA+ current that displayed faster decay during ATP stimulation than mP2X7R alone. Moreover, cells transfected with a 2 : 1 cDNA ratio displayed decaying kinetics similar to those observed in acinar cells. Concentration–response curves in Na+-containing solutions were constructed for heterologously expressed mP2X4R, mP2X7R and mP2X4R:mP2X7R co-expressions as well as acinar cells. The EC50 values determined were 11, 220, 434 and 442 μm, respectively. Na+ currents generated by expressing mP2X4R or mP2X7R alone were potentiated by ivermectin (IVM). In contrast, IVM potentiation in acinar cells and HEK cells co-expressing P2X4 and P2X7 (1 : 1 or 2 : 1 cDNA ratios) was seen only when the ATP concentration was lowered from 5 to 0.03 mm. Taken together our observations indicate a functional interaction between murine P2X7 and P2X4 receptors. Such interaction might occur in acinar cells to shape the response to extracellular ATP in salivary epithelia.