Ethanol potently and competitively inhibits binding of the alcohol antagonist Ro15-4513 to α4/6β3δ GABAA receptors

Abstract

Although GABAA receptors have long been implicated in mediating ethanol (EtOH) actions, receptors containing the "nonsynaptic" δ subunit only recently have been shown to be uniquely sensitive to EtOH. Here, we show that δ subunit-containing receptors bind the imidazo- benzodiazepines (BZs) flumazenil and Ro15-4513 with high affinity (Kd < 10 nM), contrary to the widely held belief that these receptors are insensitive to BZs. In immunopurified native cerebellar and recombinant δ subunit-containing receptors, binding of the alcohol antagonist [ 3H]Ro15-4513 is inhibited by low concentrations of EtOH (K i ≈ 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, β-carbolinecarboxylate ethyl ester (β-CCE), and N-methyl-β-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. © 2006 by The National Academy of Sciences of the USA.