Molecular requirements for ethanol differential allosteric modulation of glycine receptors based on selective Gbetagamma modulation.

It is now believed that the allosteric modulation produced by ethanol in glycine receptors (GlyRs) depends on alcohol binding to discrete sites within the protein structure. Thus, the differential ethanol sensitivity of diverse GlyR isoforms and mutants was explained by the presence of specific residues in putative alcohol pockets. Here, we demonstrate that ethanol sensitivity in two ligand-gated ion receptor members, the GlyR adult alpha(1) and embryonic alpha(2) subunits, can be modified through selective mutations that rescued or impaired Gbetagamma modulation. Even though both isoforms were able to physically interact with Gbetagamma, only the alpha(1) GlyR was functionally modulated by Gbetagamma and pharmacological ethanol concentrations. Remarkably, the simultaneous switching of two transmembrane and a single extracellular residue in alpha(2) GlyRs was enough to generate GlyRs modulated by Gbetagamma and low ethanol concentrations. Interestingly, although we found that these TM residues were different to those in the alcohol binding site, the extracellular residue was recently implicated in conformational changes important to generate a pre-open-activated state that precedes ion channel gating. Thus, these results support the idea that the differential ethanol sensitivity of these two GlyR isoforms rests on conformational changes in transmembrane and extracellular residues within the ion channel structure rather than in differences in alcohol binding pockets. Our results describe the molecular basis for the differential ethanol sensitivity of two ligand-gated ion receptor members based on selective Gbetagamma modulation and provide a new mechanistic framework for allosteric modulations of abuse drugs.