Low dose acute alcohol effects on GABA A receptor subtypes.

GABA(A) receptors (GABA(A)Rs) are the main inhibitory neurotransmitter receptors and have long been implicated in mediating at least part of the acute actions of ethanol. For example, ethanol and GABAergic drugs including barbiturates and benzodiazepines share many pharmacological properties. Besides the prototypical synaptic GABA(A)R subtypes, nonsynaptic GABA(A)Rs have recently emerged as important regulators of neuronal excitability. While high doses (> or =100 mM) of ethanol have been reported to enhance activity of most GABA(A)R subtypes, most abundant synaptic GABA(A)Rs are essentially insensitive to ethanol concentrations that occur during social ethanol consumption (< 30 mM). However, extrasynaptic delta and beta3 subunit-containing GABA(A)Rs, associated in the brain with alpha4 or alpha6 subunits, are sensitive to low millimolar ethanol concentrations, as produced by drinking half a glass of wine. Additionally, we found that a mutation in the cerebellar alpha6 subunit (alpha6R100Q), initially reported in rats selectively bred for increased alcohol sensitivity, is sufficient to produce increased alcohol-induced motor impairment and further increases of alcohol sensitivity in recombinant alpha6beta3delta receptors. Furthermore, the behavioral alcohol antagonist Ro15-4513 blocks the low dose alcohol enhancement on alpha4/6/beta3delta receptors, without reducing GABA-induced currents. In binding assays alpha4beta3delta GABA(A)Rs bind [(3)H]Ro15-4513 with high affinity, and this binding is inhibited, in an apparently competitive fashion, by low ethanol concentrations, as well as analogs of Ro15-4513 that are active to antagonize ethanol or Ro15-4513's block of ethanol. We conclude that most low to moderate dose alcohol effects are mediated by alcohol actions on alcohol/Ro15-4513 binding sites on GABA(A)R subtypes.

Ethanol potently and competitively inhibits binding of the alcohol antagonist Ro15-4513 to alpha4/6beta3delta GABAA receptors.

Although GABA(A) receptors have long been implicated in mediating ethanol (EtOH) actions, receptors containing the "nonsynaptic" delta subunit only recently have been shown to be uniquely sensitive to EtOH. Here, we show that delta subunit-containing receptors bind the imidazo-benzodiazepines (BZs) flumazenil and Ro15-4513 with high affinity (K(d) < 10 nM), contrary to the widely held belief that these receptors are insensitive to BZs. In immunopurified native cerebellar and recombinant delta subunit-containing receptors, binding of the alcohol antagonist [(3)H]Ro15-4513 is inhibited by low concentrations of EtOH (K(i) approximately 8 mM). Also, Ro15-4513 binding is inhibited by BZ-site ligands that have been shown to reverse the behavioral alcohol antagonism of Ro15-4513 (i.e., flumazenil, beta-carbolinecarboxylate ethyl ester (beta-CCE), and N-methyl-beta-carboline-3-carboxamide (FG7142), but not including any classical BZ agonists like diazepam). Experiments that were designed to distinguish between a competitive and allosteric mechanism suggest that EtOH and Ro15-4513 occupy a mutually exclusive binding site. The fact that only Ro15-4513, but not flumazenil, can inhibit the EtOH effect, and that Ro15-4513 differs from flumazenil by only a single group in the molecule (an azido group at the C7 position of the BZ ring) suggest that this azido group in Ro15-4513 might be the area that overlaps with the alcohol-binding site. Our findings, combined with previous observations that Ro15-4513 is a behavioral alcohol antagonist, suggest that many of the behavioral effects of EtOH at relevant physiological concentrations are mediated by EtOH/Ro15-4513-sensitive GABA(A) receptors.

Contributions of the GABAA receptor alpha6 subunit to phasic and tonic inhibition revealed by a naturally occurring polymorphism in the alpha6 gene.

GABAA receptors (GABARs) are heteromultimeric proteins composed of five subunits. The specific subunit composition determines critical properties of a GABAR such as pharmacological sensitivities and whether the receptor contributes to synaptic or extrasynaptic forms of inhibition. Classically, synaptic but not extrasynaptic GABARs are thought to respond to benzodiazepines, whereas the reverse has been suggested for ethanol. To examine the effects of subunit composition on GABAR function in situ, we took advantage of two naturally occurring alleles of the rat gene for GABAR subunit alpha6 (Gabra6(100R) and Gabra6(100Q)). Depending on their subunit partners, these two variants of alpha6 can lead to differential sensitivities to benzodiazepines and ethanol. An examination of synaptic and extrasynaptic GABA-mediated currents in cerebellar granule cells from Gabra6(100R/100R) and Gabra6(100Q/100Q) rats uncovered marked allele-dependent differences in benzodiazepine sensitivity. Unexpectedly, we found that the benzodiazepines flunitrazepam and diazepam enhanced extrasynaptic inhibition mediated by delta subunit-containing GABARs in Gabra6(100Q/100Q) rats. Complementary experiments on recombinant GABARs confirmed that, at subsaturating [GABA], flunitrazepam potentiates alpha6/delta subunit-containing GABARs. Based on data and a simple theoretical analysis, we estimate that the average extrasynaptic [GABA] is approximately 160 nm in perfused slices. These results (1) demonstrate contributions of alpha6 subunits to both synaptic and extrasynaptic GABA responses, (2) establish that delta subunit-containing GABARs are benzodiazepine sensitive at subsaturating [GABA] and, (3) provide an empirical estimate of extrasynaptic [GABA] in slices.

Alcohol-induced motor impairment caused by increased extrasynaptic GABA(A) receptor activity.

Neuronal mechanisms underlying alcohol intoxication are unclear. We find that alcohol impairs motor coordination by enhancing tonic inhibition mediated by a specific subtype of extrasynaptic GABA(A) receptor (GABAR), alpha6beta3delta, expressed exclusively in cerebellar granule cells. In recombinant studies, we characterize a naturally occurring single-nucleotide polymorphism that causes a single amino acid change (R100Q) in alpha6 (encoded in rats by the Gabra6 gene). We show that this change selectively increases alcohol sensitivity of alpha6beta3delta GABARs. Behavioral and electrophysiological comparisons of Gabra6(100R/100R) and Gabra6(100Q/100Q) rats strongly suggest that alcohol impairs motor coordination by enhancing granule cell tonic inhibition. These findings identify extrasynaptic GABARs as critical targets underlying low-dose alcohol intoxication and demonstrate that subtle changes in tonic inhibition in one class of neurons can alter behavior.

Alcohol effects on gamma-aminobutyric acid type A receptors: are extrasynaptic receptors the answer?

GABA(A) receptors have long been implicated in mediating at least part of the actions of ethanol in mammalian brain. However, until very recently, reports of the actions of EtOH on recombinant receptors have required very high doses of ethanol and animals lacking receptor subunits shown to be important for ethanol actions in vitro did not support the view that these subunits are crucial in ethanol actions. Recombinant alpha4beta3delta and alpha6beta3delta GABA(A) receptors are uniquely sensitive to ethanol, with a dose-response relationship mirroring the well known effects of alcohol consumption on the human brain. Receptors containing the delta subunit are thought to be located extrasynaptically and it will be important to determine if these extrasynaptic GABA(A) receptor subunit combinations mediate low dose alcohol effects in vivo.

Fishing for allosteric sites on GABA(A) receptors.

GABA(A) receptors have structural and functional homology with a super-family of cys-loop ligand-gated ion channel receptors including the nicotinic acetylcholine receptors. Amino acid residues involved in ligand-binding pockets are homologous among super-family members, leading to the multiple-loop model of binding sites situated at subunit interfaces, validated by structural studies on the nicotinic acetylcholine receptor and water-soluble snail acetylcholine binding protein. This article will briefly review the literature on the agonist binding sites on the receptor super-family, and then describe the current situation for attempts to identify sites for allosteric modulators on the GABA(A) receptors. A combination of mutagenesis and photoaffinity labeling with anesthetic ligands has given some leads in this endeavor. Current work by others and ourselves focuses on three putative sites for modulators: (1) within the ion channel domain TM2, near the extracellular end; (2) the agonist binding sites and homologous pockets at other subunit interfaces of the pentameric receptor; and (3) on the linker region stretching from the agonist site loop C to the top of the TM1 region. It is likely that concrete structural information will be forthcoming soon.