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-bisphos-phate [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 μM. 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 mol% PI(4,5)P₂ in a phosphatidylcholine bilayer identified 8 binding sites with significant probability of binding PI(4,5)P₂. Three of these sites captured 85% of all ANO1–PI(4,5)P₂ interactions. Mutagenesis of basic amino acids near the membrane–cytosol interface found 3 regions of ANO1 critical for PI(4,5)P₂ regulation that correspond to the same 3 sites identified by MD. PI(4,5)P₂ is stabilized by hydrogen bonding between amino acid side chains 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 3 PI(4,5)P₂ binding sites at the cytoplasmic face of the membrane allosterically regulating ANO1 channel gating.