A network of phosphatidylinositol 4,5-bisphosphate binding sites regulates gating of the Ca2+-activated Cl- channel ANO1 (TMEM16A)

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)P-2]. 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)P-2 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)P-2 with an EC50 = 1.24 mu M. The effect of PI(4,5)P-2 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 mol% PI(4,5)P-2 in a phosphatidylcholine bilayer identified 8 binding sites with significant probability of binding PI(4,5)P-2. Three of these sites captured 85% of all ANO1-PI(4,5)P-2 interactions. Mutagenesis of basic amino acids near the membranecytosol interface found 3 regions of ANO1 critical for PI(4,5)P-2 regulation that correspond to the same 3 sites identified by MD. PI(4,5)P-2 is stabilized by hydrogen bonding between amino acid side chains and phosphate/hydroxyl groups on PI(4,5)P-2. Binding of PI(4,5)P-2 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 3 PI(4,5)P-2 binding sites at the cytoplasmic face of the membrane allosterically regulating ANO1 channel gating.