Kuai Yu, Tao Jiang, Yuanyuan Cui, Emad Tajkhorshid, and H Criss Hartzell.
A network of phosphatidylinositol 4,5-bisphosphate binding sites
regulate gating of the Ca2+-activated Cl− channel ANO1
(TMEM16A).
Proceedings of the National Academy of Sciences, USA,
116:19952-19962, 2019.
(PMC: PMC6778221)
YU2019-ET
ANO1 (TMEM16A) is a Ca2+ activated Cl- channel that regulates diverse cellular
functions including fluid
secretion, neuronal excitability, and smooth muscle contraction. ANO1 is
activated by elevation of
cytosolic Ca2+ and modulated by phosphatidylinositol 4,5-bisphosphate
(PI(4,5)P2). Here we describe a
closely concerted experimental and computational study, including
electrophysiology, mutagenesis,
functional assays, and extended sampling of lipid-protein interactions with
molecular dynamics (MD) to
characterize PI(4,5)P2 binding modes and sites on ANO1. ANO1 currents in
excised inside-out patches
activated by 270 nM Ca2+ at +100 mV are increased by exogenous PI(4,5)P2
with an EC50 = 1.24 . The
effect of PI(4,5)P2 is dependent on membrane voltage and Ca2+ and is
explained by a stabilization of
the ANO1 Ca2+ bound open state. Unbiased atomistic MD simulations with 1.4
molphosphatidylcholine bilayer identified 8 binding sites with significant probability
of binding PI(4,5)P2.
Three of these sites captured 85Mutagenesis of basic amino
acids near the membrane-cytosol interface found three regions of ANO1 critical
for PI(4,5)P2 regulation
that correspond to the same three sites identified by MD. PI(4,5)P2 is stabilized
by hydrogen bonding
between amino acid sidechains and phosphate/hydroxyl groups on PI(4,5)P2.
Binding of PI(4,5)P2 alters
the position of the cytoplasmic extension of TM6, which plays a crucial role in
ANO1 channel gating, and
increases the accessibility of the inner vestibule to Cl- ions. We propose a
model consisting of a network
of three PI(4,5)P2 binding sites at the cytoplasmic face of the membrane
allosterically regulating ANO1
channel gating.
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