Gating modes of calcium-activated chloride channels TMEM16A and TMEM16B

摘要

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 ([Ca2+](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 mu M 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.