A Network of Phosphatidylinositol 4,5-bisphosphate Binding Sites Regulate Gating of the Ca 2+ -activated Cl − Channel ANO1 (TMEM16A)

ANO1 (TMEM16A) is a Ca 2+ -activated Cl − channel that regulates diverse cellular functions including fluid secretion, neuronal excitability, and smooth muscle contraction. ANO1 is activated by elevation of cytosolic Ca 2+ 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 Ca 2+ at +100 mV are increased by exogenous PI(4,5)P 2 with an EC 50 = 1.24 µM. The effect of PI(4,5)P 2 is dependent on membrane voltage and Ca 2+ and is explained by a stabilization of the ANO1 Ca 2+ -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 membrane-cytosol interface found three regions of ANO1 critical for PI(4,5)P 2 regulation that correspond to the same three sites identified by MD. PI(4,5)P 2 is stabilized by hydrogen bonding between amino acid sidechains 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 three PI(4,5)P 2 binding sites at the cytoplasmic face of the membrane allosterically regulating ANO1 channel gating. Significance statement Membrane proteins dwell in a sea of phospholipids that not only structurally stabilize the proteins by providing a hydrophobic environment for their transmembrane segments, but also dynamically regulate protein function. While many cation channels are known to be regulated by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ), relatively little is known about anion channel regulation by phosphoinositides. Using a combination of patch clamp electrophysiology and atomistic molecular dynamics simulations, we have identified several PI(4,5)P 2 binding sites in ANO1 (TMEM16A), a Cl − channel that performs myriad physiological functions from epithelial fluid secretion to regulation of electrical excitability. These binding sites form a band at the cytosolic interface of the membrane that we propose constitute a network to dynamically regulate this highly allosteric protein.