Low-dose alcohol actions on α4β3δ GABAA receptors are reversed by the behavioral alcohol antagonist Ro15-4513

Although it is now more than two decades since it was first reported that the imidazobenzodiazepine Ro15-4513 reverses behavioral alcohol effects, the molecular target(s) of Ro15-4513 and the mechanism of alcohol antagonism remain elusive. Here, we show that Ro15-45113 blocks the alcohol enhancement on recombinant "extrasynaptic" alpha 4/6 beta 3 delta GABA(A) receptors at doses that do not reduce the GABA-incluced Cl- current. At low ethanol concentrations (<= 30 mM), the Ro15-4513 antagonism is complete. However, at higher ethanol concentrations (>= 1100 mM), there is a Ro15-4513-insensitive ethanol enhancement that is abolished in receptors containing a point mutation in the second transmembrane region of the 133 subunit (beta 3N265M). Therefore, alpha 4/6 beta 3 delta GABA receptors have two distinct alcohol modulation sites: (i) a low-dose ethanol site present in alpha 4/6 beta 3 delta receptors that is antagonized by the behavioral alcohol antagonist Ro15-45113 and (h) a site activated at high (anesthetic) alcohol doses, defined by mutations in membrane-spanning regions. Receptors composed of alpha 4 beta 3N265M delta subunits that lack the high-dose alcohol site show a saturable ethanol dose-response curve with a half-maximal enhancement at 16 mM, close to the legal blood alcohol driving limit in most U.S. states (17.4 mM). Like in behavioral experiments, the alcohol antagonist effect of Ro15-4513 on recombinant alpha 4 beta 3 delta receptors is blocked by flumazenil and beta-carboline-ethyl ester (beta-CCE). Our findings suggest that ethanol/Ro15-4513-sensitive GABA(A) receptors are important mediators of behavioral alcohol effects.