Multiple Modes of Interaction between the Nicotinic Acetylcholine Receptor and Isoflurane Observed Through Long Time Simulations

Abstract

Understanding of the molecular mechanisms through which anesthetics affect function of ligand gated ion channels (LGIC) like the nicotinic acetylcholine receptor (nAChR) is complicated by the multitude of binding sites and possible mechanisms that are consistent with experimental data, including pore-block, competitive binding, and allosteric mechanisms. Molecular dynamics (MD) studies of general anesthetics and Cys-loop receptors have been hindered by low resolution and unstable structures and the computational cost of simulating large systems for the long times required to observe all binding modes. We present long time (400 ns) simulations in which isoflurane introduced into the water surrounding the nAChR proceeded to bind to four main classes of site on the protein (as well as partition into the membrane) : 1) the hydrophobic constriction of the channel lumen, effectively blocking the pore 2) the agonist site 3) the interface between subunits, in the transmembrane domain, 4) in the center of some subunits, in the transmembrane domain on the intracellular half. Sites 1) and 2) are most likely inhibitory sites that may not be present in anionic cys-loop channels (which are potentiated by anesthetics);we find analogous sites in a 200 ns simulation of the prokaryotic cationic LGIC from Gloeobacter violaceus (for which two high resolution structures are available). Isoflurane bound to site 3) is found to have distinct and statistically significant effects on the structure and dynamics of the adjacent critical M2-M3 loop, with repercussions in the pore-lining M2 helices,consistent with an allosteric potentiating mechanism that may account for the reverse effect of isoflurane on the GABA and glycine receptors.