Control of Ethanol Sensitivity of the Glycine Receptor α3 Subunit by Transmembrane 2, the Intracellular Splice Cassette and C-Terminal Domain

Previous studies have shown that the effect of ethanol on glycine receptors (GlyRs) containing the α 1 subunit is affected by interaction with heterotrimeric G proteins (G βγ ). GlyRs containing the α 3 subunit are involved in inflammatory pain sensitization and rhythmic breathing and have received much recent attention. For example, it is unknown whether ethanol affects the function of this important GlyR subtype. Electrophysiologic experiments showed that GlyR α 3 subunits were not potentiated by pharmacologic concentrations of ethanol or by G βγ . Thus, we studied GlyR α 1– α 3 chimeras and mutants to determine the molecular properties that confer ethanol insensitivity. Mutation of corresponding glycine 254 in transmembrane domain 2 (TM2) found in α 1 in the α 3 A254G – α 1 chimera induced a glycine-evoked current that displayed potentiation during application of ethanol (46 ± 5%, 100 mM) and G βγ activation (80 ± 17%). Interestingly, insertion of the intracellular α 3L splice cassette into GlyR α 1 abolished the enhancement of the glycine-activated current by ethanol (5 ± 6%) and activation by G βγ (−1 ± 7%). Incorporation of the GlyR α 1 C terminus into the ethanol-resistant α 3S A254G mutant produced a construct that displayed potentiation of the glycine-activated current with 100 mM ethanol (40 ± 6%) together with a current enhancement after G protein activation (68 ± 25%). Taken together, these data demonstrate that GlyR α 3 subunits are not modulated by ethanol. Residue A254 in TM2, the α 3L splice cassette, and the C-terminal domain of α 3 GlyRs are determinants for low ethanol sensitivity and form the molecular basis of subtype-selective modulation of GlyRs by alcohol.