Molecular Mechanism for the Dual Alcohol Modulation of Cys-loop Receptors

Cys-loop receptors constitute a superfamily of pentameric ligand-gated ion channels (pLGICs), including receptors for acetylcholine, serotonin, glycine and γ-aminobutyric acid. Several bacterial homologues have been identified that are excellent models for understanding allosteric binding of alcohols and anesthetics in human Cys-loop receptors. Recently, we showed that a single point mutation on a prokaryotic homologue (GLIC) could transform it from a channel weakly potentiated by ethanol into a highly ethanol-sensitive channel. Here, we have employed molecular simulations to study ethanol binding to GLIC, and to elucidate the role of the ethanol-enhancing mutation in GLIC modulation. By performing 1-µs simulations with and without ethanol on wild-type and mutated GLIC, we observed spontaneous binding in both intra-subunit and inter-subunit transmembrane cavities. In contrast to the glycine receptor GlyR, in which we previously observed ethanol binding primarily in an inter-subunit cavity, ethanol primarily occupied an intra-subunit cavity in wild-type GLIC. However, the highly ethanol-sensitive GLIC mutation significantly enhanced ethanol binding in the inter-subunit cavity. These results demonstrate dramatic effects of the F(14′)A mutation on the distribution of ligands, and are consistent with a two-site model of pLGIC inhibition and potentiation. Communication from one nerve cell to the next is an essential process for brain and muscle function. Nerve impulses result in release of transmitter molecules from one cell that bind to receptors on the next cell. Transmitter binding opens a pore in each receptor and ions flow across the membrane, leading to either enhancement or inhibition of new nerve impulses. These receptors are modulated by numerous drugs, including alcohols and anesthetics; identifying the precise location of modulator binding is critical for drug development. We have used computer simulation methods to model alcohol diffusion and binding to a receptor. By modifying a single residue in the receptor, we were able to move the location of the binding site and dramatically alter alcohol modulation, which supports a model with two separate binding sites for enhancement and inhibition in this family of receptors.