摘要：Anoctamin-1 (ANO1 or TMEM16A) is a homo-dimeric Ca2 -activated Cl− channel responsible for essential physiological processes. Each monomer harbours a pore and a Ca2 -binding pocket; the voltage-dependent binding of two intracellular Ca2 ions to the pocket gates the pore. However, in the absence of intracellular Ca2 voltage activates TMEM16A by an unknown mechanism. Here we show voltage-activated anion currents that are outwardly rectifying, time-independent with fast or absent tail currents that are inhibited by tannic and anthracene-9-carboxylic acids. Since intracellular protons compete with Ca2 for binding sites in the pocket, we hypothesized that voltage-dependent titration of these sites would induce gating. Indeed intracellular acidification enabled activation of TMEM16A by voltage-dependent protonation, which enhanced the open probability of the channel. Mutating Glu/Asp residues in the Ca2 -binding pocket to glutamine (to resemble a permanent protonated Glu) yielded channels that were easier to activate at physiological pH. Notably, the response of these mutants to intracellular acidification was diminished and became voltage-independent. Thus, voltage-dependent protonation of glutamate/aspartate residues (Glu/Asp) located in the Ca2 -binding pocket underlines TMEM16A activation in the absence of intracellular Ca2 .
