Molecular underpinning of intracellular pH regulation on TMEM16F

TMEM16F, a dual-function phospholipid scramblase and ion channel, is important in blood coagulation, skeleton development, HIV infection, and cell fusion. Despite advances in understanding its structure and activation mechanism, how TMEM16F is regulated by intracellular factors remains largely elusive. Here we report that TMEM16F lipid scrambling and ion channel activities are strongly influenced by intracellular pH (pH(i)). We found that low pH(i) attenuates, whereas high pH(i) potentiates, TMEM16F channel and scramblase activation under physiological concentrations of intracellular Ca(2+) ([Ca(2+)](i)). We further demonstrate that TMEM16F pH(i) sensitivity depends on [Ca(2+)](i) and exhibits a bell-shaped relationship with [Ca(2+)](i): TMEM16F channel activation becomes increasingly pH(i) sensitive from resting [Ca(2+)](i) to micromolar [Ca(2+)](i), but when [Ca(2+)](i) increases beyond 15 µM, pH(i) sensitivity gradually diminishes. The mutation of a Ca(2+)-binding residue that markedly reduces TMEM16F Ca(2+) sensitivity (E667Q) maintains the bell-shaped relationship between pH(i) sensitivity and Ca(2+) but causes a dramatic shift of the peak [Ca(2+)](i) from 15 µM to 3 mM. Our biophysical characterizations thus pinpoint that the pH(i) regulatory effects on TMEM16F stem from the competition between Ca(2+) and protons for the primary Ca(2+)-binding residues in the pore. Within the physiological [Ca(2+)](i) range, the protonation state of the primary Ca(2+)-binding sites influences Ca(2+) binding and regulates TMEM16F activation. Our findings thus uncover a regulatory mechanism of TMEM16F by pH(i) and shine light on our understanding of the pathophysiological roles of TMEM16F in diseases with dysregulated pH(i), including cancer.