Low-dose alcohol actions on 4 3 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 Ro15-4513 blocks the alcohol enhancement on recombinant ‘‘extrasynaptic’’ 4 6 3 GABAA receptors at doses that do not reduce the GABA-induced Cl current. At low ethanol concentrations (<30 mM), the Ro15-4513 antagonism is complete. However, at higher ethanol concentrations (>100 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 3 subunit ( 3N265M). Therefore, 4 6 3 GABA receptors have two distinct alcohol modulation sites: (i) a low-dose ethanol site present in 4 6 3 receptors that is antagonized by the behavioral alcohol antagonist Ro15-4513 and (ii) a site activated at high (anesthetic) alcohol doses, defined by mutations in membrane-spanning regions. Receptors composed of 4 3N265M 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 4 3 receptors is blocked by flumazenil and -carboline-ethyl ester ( -CCE). Our findings suggest that ethanol Ro15-4513-sensitive GABAA receptors are important mediators of behavioral alcohol effects.