A Network of Phosphatidylinositol 4,5-bisphosphate Binding Sites Regulate Gating of the Ca²⁺-activated Cl⁻ Channel ANO1 (TMEM16A)

Abstract

ANO1 (TMEM16A) is a Ca 2+ -activated Clchannel 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)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 Ca 2+ at +100 mV are increased by exogenous PI(4,5)P2with an EC50 = 1.24 µM. The effect of PI(4,5)P2 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% 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)P2interactions. Mutagenesis 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 Clions. 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.of the authors and does not necessarily represent the official views of the National Institutes of Health. We also acknowledge computing resources provided by Blue Waters at National Center for Supercomputing Applications, and Extreme Science and Engineering Discovery Environment (grant MCA06N060 to ET).

Binding mode of SARS-CoV-2 fusion peptide to human cellular membrane

“…As shown in previous studies, compared with conventional (full) membrane simulations, physical characteristics of membrane binding remain conserved for peripheral proteins, but the binding timescale is reduced by an order of magnitude with the use of HMMM (38,41,55). The HMMM model has been extensively used in a broad spectrum of systems providing mechanistic details of lipid-protein interactions (37)(38)(39)(40)(41)(43)(44)(45)(46)(47)(48)(49)(56)(57)(58)(59)(60)(61)(62)(63)(64)(65) and can accurately reproduce the energetics associated with partitioning of amino acids within the headgroups and in the most peripheral section of the leaflets (35). We use this technique to facilitate binding and insertion of the FP into the membrane, which would allow us to focus our computational effort on running many independent membrane-binding simulations.…”

Binding Mode of SARS-CoV2 Fusion Peptide to Human Cellular Membrane

“…timescale is reduced by an order of magnitude with the use of HMMM. 38,41,55 The HMMM model has been extensively used in a broad spectrum of systems providing mechanistic details of lipid-protein interactions [37][38][39][40][41][43][44][45][46][47][48][49][56][57][58][59][60][61][62][63][64][65] and can accurately reproduce the energetics associated with partitioning of amino acids within the head groups and in the most peripheral section of the leaflets. 35 We use this technique to facilitate binding and insertion of the FP into the membrane, which would allow us to focus our computational effort on running many independent membrane-binding simulations.…”

Phosphate position is key in mediating transmembrane ion channel TMEM16A–phosphatidylinositol 4,5-bisphosphate interaction

“…In addition to intracellular Ca 2+ , TMEM16A also requires the acidic phospholipid phosphatidylinositol 4,5bisphosphate (PI(4,5)P 2 ) in order to transition to the open conformation (25)(26)(27)(28)(29). Like the currents of other channels potentiated by PI(4,5)P 2 (30), TMEM16A currents recorded using the inside-out configuration of the patch-clamp technique rundown over time, even with continued application of intracellular Ca 2+ .…”