TCB Publications - Abstract

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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