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Ethanol increased the frequency of miniature glycinergic currents [miniature inhibitory postsynaptic currents (mIPSCs)] in cultured spinal neurons. This effect was dependent on intracellular calcium augmentation, since preincubation with BAPTA (an intracellular calcium chelator) or thapsigargin [a sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) pump inhibitor] significantly attenuated this effect. Similarly, U73122 (a phospholipase C inhibitor) or 2-aminoethoxydiphenyl borate [2-APB, an inositol 1,4,5-trisphosphate (IP₃) receptor (IP3R) inhibitor] reduced this effect. Block of ethanol action was also achieved after preincubation with Rp-cAMPS, inhibitor of the adenylate cyclase (AC)/PKA signaling pathway. These data suggest that there is a convergence at the level of IP₃R that accounts for presynaptic ethanol effects. At the postsynaptic level, ethanol increased the decay time constant of mIPSCs in a group of neurons (30 ± 10% above control, n = 13/26 cells). On the other hand, the currents activated by exogenously applied glycine were consistently potentiated (55 ± 10% above control, n = 11/12 cells), which suggests that ethanol modulates synaptic and nonsynaptic glycine receptors (GlyRs) in a different fashion. Supporting the role of G protein modulation on ethanol responses, we found that a nonhydrolyzable GTP analog [guanosine 5'-O-(3-thiotriphosphate) (GTPγS)] increased the decay time constant in ∼50% of the neurons (28 ± 12%, n = 11/19 cells) but potentiated the glycine-activated Cl(-) current in most of the neurons examined (83 ± 29%, n = 7/9 cells). In addition, confocal microscopy showed that α1-containing GlyRs colocalized with Gβ and Piccolo (a presynaptic cytomatrix protein) in ∼40% of synaptic receptor clusters, suggesting that colocalization of Gβγ and GlyRs might account for the difference in ethanol sensitivity at the postsynaptic level.

Previous studies have shown that the effect of ethanol on glycine receptors (GlyRs) containing the a1 subunit is affected by interaction with heterotrimeric G proteins (Gbg). GlyRs containing the a3 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 a3 subunits were not potentiated by pharmacologic concentrations of ethanol or by Gbg. Thus, we studied GlyR a1-a3 chimeras and mutants to determine the molecular properties that confer ethanol insensitivity. Mutation of corresponding glycine 254 in transmembrane domain 2 (TM2) found in a1 in the a3 A254G -a1 chimera induced a glycine-evoked current that displayed potentiation during application of ethanol (46 6 5%, 100 mM) and Gbg activation (80 6 17%). Interestingly, insertion of the intracellular a3L splice cassette into GlyR a1 abolished the enhancement of the glycine-activated current by ethanol (5 6 6%) and activation by Gbg (21 6 7%). Incorporation of the GlyR a1 C terminus into the ethanol-resistant a3S A254G mutant produced a construct that displayed potentiation of the glycine-activated current with 100 mM ethanol (40 6 6%) together with a current enhancement after G protein activation (68 6 25%). Taken together, these data demonstrate that GlyR a3 subunits are not modulated by ethanol. Residue A254 in TM2, the a3L splice cassette, and the C-terminal domain of a3 GlyRs are determinants for low ethanol sensitivity and form the molecular basis of subtype-selective modulation of GlyRs by alcohol.

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