期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2019
卷号：116
期号：40
页码：19952-19962
DOI：10.1073/pnas.1904012116
出版社：The National Academy of Sciences of the United States of America
摘要：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 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..
