Abstract
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 ([Ca(2+) ]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 480 RSQ482 to AVK in the first intracellular loop of TMEM16B nearly abolished slow gating, but, mutating 448 AVK451 to RSQ in TMEM16A has little effect. Deleting 448 EAVK451 residues in TMEM16A reduced slow gating. We conclude that TMEM16 CaCCs have intrinsic Vm - and Cl(-) -sensitive dual gating that elicits complex ICl kinetics.