Molecular underpinning of intracellular pH regulation on TMEM16F

TMEM16F, a dual functional 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 (pHi).
We found that low pHi attenuates, whereas high pHi potentiates, TMEM16F
channel and scramblase activation under physiological concentrations of
intracellular Ca2+ ([Ca2+]i). We further demonstrate that TMEM16F pHi
sensitivity depends on [Ca2+]i and exhibits a bell-shaped relationship
with [Ca2+]i: TMEM16F channel activation becomes increasingly pHi
sensitive from the resting [Ca2+]i to micromolar [Ca2+]i, but when [Ca2+]i
increases beyond 15µM, the pHi sensitivity gradually diminishes. The
mutation of a Ca2+-binding residue that markedly reduces TMEM16F Ca2+
sensitivity (E667Q), maintains the bell-shaped relationship between pHi
sensitivity and Ca2+ but causes a dramatic shift of the peak [Ca2+]i from
15µM to 3 mM. Our biophysical characterizations thus pinpoints that the
pHi regulatory effects on TMEM16F stem from the competition between Ca2+
and proton for the primary Ca2+-binding residues in the pore. Within the
physiological [Ca2+]i range, the protonation state of the primary
Ca2+-binding sites influences Ca2+ binding and regulates TMEM16F
activation. Our findings thus uncover a regulatory mechanism of TMEM16F by
pHi and shine light on our understanding of the pathophysiological roles
of TMEM16F in diseases with dysregulated pHi including cancer.