The Molecular Mechanism for the Dual Alcohol Modulation of Cys-Loop Receptors

Abstract

Despite high sequence identity, pentameric ligand-gated ion channels exhibit remarkable diversity in function with anionic/cationic channels that are either potentiated or inhibited by allosteric ligands. The recently available structures of bacterial homologs of Cys-loop receptors provide an excellent framework for understanding this allosteric modulation and function, but the modeling can be complex; our earlier simulations of the prokaryotic anionic glycine receptor (GlyR) suggest inter-subunit binding for ethanol (Murail, Biophys J 100, 1642, 2011), which at first sight appears to be incompatible with the experimental Gloeobacter violaceus (GLIC) ligand-gated ion channel structure showing binding intra-subunit. Here, we present new simulations of GLIC that confirm the occurrence of multiple binding sites by showing intra-subunit binding for ethanol. By experimentally introducing the single-site F238A mutation in GLIC we can turn it into a highly ethanol-sensitive channel (Howard, PNAS 108, 12149, 2011), similar to GlyR, and simulations of the mutated species confirm the occurrence of multiple binding sites. To critically test the results, we performed extensive docking and free energy calculations to identify alcohol-binding sites and determine their affinity. In the wild-type GLIC, short alcohols preferentially bind intra-subunit, with a very weak binding site inter-subunit. However, with the F238A mutation the inter-subunit site achieves a significantly lower free energy, and even becomes the highest-affinity site in the channel for some alcohols. These results suggest a new model for pentameric ligand-gated channel potentiation and inhibition, where the intra-subunit cavity would control inhibition, and the inter-subunit cavity potentiation. The possibility of allosteric ligands interacting with both cavities - or even a single molecule stretching from one site to the other - offers an attractive explanation for the complex functional dynamics of ligand-gated ion channels as well as an potential explain for the alcohol cutoff effect.