There is substantial evidence that GABAergic neurotransmission is important for many behavioral actions of ethanol and there are reports spanning more than 30 years of literature showing that low to moderate (3-30 mM) concentrations of ethanol enhance GABAergic neurotransmission. A key question is which GABA receptor subunits are sensitive to low concentrations of ethanol in vivo and in vitro. Recent evidence points to a role for extrasynaptic receptors. Another question is which behavioral actions of alcohol result from enhancement of GABAergic neurotransmission. Some clues are beginning to emerge from studies of knock-out and knock-in mice and from genetic analysis of human alcoholics. These approaches are converging on a role for GABAergic actions in regulating alcohol consumption and, perhaps, the development of alcoholism.

The glycine receptor is a target for both alcohols and anesthetics, and certain amino acids in the ␣1 subunit transmembrane segments (TM) are critical for drug effects. Introducing larger amino acids at these positions increases the potency of glycine, suggesting that introducing larger residues, or drug molecules, into the drugbinding cavity facilitates channel opening. A possible mechanism for these actions is that the volume of the cavity expands and contracts during channel opening and closing. To investigate this hypothesis, mutations for amino acids in TM1 (I229C) and TM2 (G256C, T259C, V260C, M263C, T264C, and A288C showed state-dependent reaction with MTS only in the presence of agonist. M263C and S270C were also accessible to MTS labeling. Mutated residues more intracellular than M263C did not react, indicating a floor of the cavity. These data demonstrate that the conformational changes accompanying channel gating increase accessibility to amino acids critical for drug action in TM1, TM2, and TM3, which may provide a mechanism by which alcohols and anesthetics can act on glycine (and likely other) receptors.The glycine receptor (GlyR) 1 is a target for both alcohols and anesthetics. Three amino acids were previously identified as critical for alcohol and/or volatile anesthetic action on glycine receptors (as well as the homologous residues on GABA A receptors): Ile 229 (in TM1), Ser 267 (in TM2), and Ala 288 (in TM3)(1-8). To study and identify water accessible residues of ion channels, such as those in drug binding pockets, methanethiosulfonate (MTS) reagents may be used as structural probes using the substituted cysteine accessibility method (9). MTS reagents rapidly react to form disulfide bonds with cysteines in the presence of water, and an irreversible change in receptor function is taken as evidence of disulfide bond formation. By use of this method, residues accessible in the presence and/or absence of neurotransmitter to sulfhydryl-specific reagents have been determined for TM2 in GABA A and acetylcholine receptors and for TM3 and the TM2-TM3 loop for GABA A receptors (10 -14 (19) found that covalent reaction of propyl methanethiosulfonate with a cysteine introduced in the putative alcohol/anesthetic binding site of the glycine receptor irreversibly enhanced receptor function and abolished further potentiation by alcohols and anesthetics (19). Glycine receptors predominate in the spinal cord and brain stem and are present in the ventral tegmental area, a brain region of importance in the rewarding effects of alcohol (20 -24). Clinically relevant concentrations of ethanol, longer chain alcohols, and volatile anesthetics enhance the function of the glycine receptor (and the homologous GABA A receptor) in heterologous expression systems (25)(26)(27). Numerous studies have shown ethanol potentiation of glycine activated currents in cultured cells, including neurons of the hippocampus and ventral tegmental area, brain synaptoneurosomes, and mouse and chick spinal cord neurons (21, 24, 28 -31)....

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Contact points between transmembrane segments (TMs) two and three of the glycine receptor are undefined and may play an important role in channel gating. We tested whether two amino acids in TM2 (S267) and TM3 (A288), known to be critical for alcohol and volatile anesthetic action, could crosslink by mutating both to cysteines and expressing the receptors in Xenopus laevis oocytes. In contrast with the wild-type receptor and single cysteine mutants, the S267C/A288C double mutant displayed unusual responses, including a tonic leak activity that was closed by strychnine and a run-down of the response upon repeated applications of glycine. We hypothesized that these characteristics were due to crosslinking of the two cysteines on opposing faces of these adjacent, alpha helical TMs. This would alter the movement of these two regions required for normal gating. To test this hypothesis, we used dithiothreitol to reduce the putative S267C-A288C disulfide bond. Reduction abolished the leak current and provided normal responses to glycine. Subsequent application of the cross-linking agent mercuric chloride caused the initial characteristics to return. These data demonstrate that S267 and A288 are near-neighbors and provide insight towards the location and role of the TM2-TM3 interface in ligand-gated ion channels.

J. Neurochem. (2008) 104, 1649–1662. Abstract The glycine receptor is a member of the Cys‐loop, ligand‐gated ion channel family and is responsible for inhibition in the CNS. We examined the orientation of amino acids I229 in transmembrane 1 (TM1) and A288 in TM3, which are both critical for alcohol and volatile anesthetic action. We mutated these two amino acids to cysteines either singly or in double mutants and expressed the receptors in Xenopus laevis oocytes. We tested whether disulfide bonds could form between A288C in TM3 paired with M227C, Y228C, I229C, or S231C in TM1. Application of cross‐linking (mercuric chloride) or oxidizing (iodine) agents had no significant effect on the glycine response of wild‐type receptors or the single mutants. In contrast, the glycine response of the I229C/A288C double mutant was diminished after application of either mercuric chloride or iodine only in the presence of glycine, indicating that channel gating causes I229C and A288C to fluctuate to be within 6 Å apart and form a disulfide bond. Molecular modeling was used to thread the glycine receptor sequence onto a nicotinic acetylcholine receptor template, further demonstrating that I229 and A288 are near‐neighbors that can cross‐link and providing evidence that these residues contribute to a single binding cavity.