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

Abstract

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'' ␣4͞6␤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, ␣4͞6␤3␦ GABA receptors have two distinct alcohol modulation sites: (i) a low-dose ethanol site present in ␣4͞6␤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 ethanol͞Ro15-4513-sensitive GABA A receptors are important mediators of behavioral alcohol effects.alcohol intoxication ͉ alcohol receptor ͉ anesthetics A lthough alcohol is one of the most widely used and abused drugs, the molecular targets that mediate alcohol effects at concentrations relevant for mild social intoxication are only beginning to be revealed. Neurotransmitter receptors for GABA, NMDA and glycine, and G protein-gated K ϩ channels have been identified as potential alcohol targets that are sensitive to intoxicating alcohol concentrations (1-4). GABA A receptors (GABA A Rs) have long been suspected to be important mediators of alcohol effects (5, 6) because benzodiazepines (BZs) and barbiturates, classic GABA A R agonists, share common pharmacological properties with ethanol, such as sedativehypnotic, anti-anxiety, and motor in-coordinating and anticonvulsant effects and have additive, possibly even synergistic effects, when taken together with ethanol (7). In addition, BZs, barbiturates, and ethanol produce tolerance and cross-tolerance to each other (8), consistent with GABA A Rs as targets of action.We have recently identified subtypes of GABA A Rs, those containing the ␦ and the ␤3 subunit, that are uniquely sensitive to low alcohol concentrations (9). Consistent with the view that ␦ subunit-containing receptors are important mediators of alcohol actions is the finding that GABA A R ␦-subunit knockout mice show multiple defects in behavioral responses to ethanol (10). Receptors containing the ␦ subunit have an exclusively nonsynaptic distribution, ...

Alcohol-sensitive GABA receptors and alcohol antagonists

“…Finally, both ␤-carboline inverse agonists, ␤-CCE and FG7142 (but not the potent inverse agonist DMCM), as well as Ro15-1788 were shown to reverse the inhibition by Ro15-4513 of alcohol-induced enhancement of ␦ subunit-containing GABA receptors, results that are strikingly reminiscent of the earlier biochemical and behavioral work (8,11). Of note, ␤-CCE actually potentiated the effects of low concentrations of ethanol (3 mM) on ␣4␤3␦ GABA A receptors and even stimulated these receptors in the absence of alcohol (3).…”

Oxytocin prevents ethanol actions at δ subunit-containing GABA A receptors and attenuates ethanol-induced motor impairment in rats

“…The δ-GABA A Rs, sometimes called the "one-glass-of-wine" receptors (24), are located in several brain regions, including the thalamus, cerebellum, hippocampus, and striatum (25,26), and show high sensitivity to relatively low doses of ethanol (2)(3)(4)(5)(6)(7)(8)27). A number of previous studies demonstrate the involvement of δ-GABA A Rs in a range of ethanolinduced functional effects, including ataxia, sedation, and reward (2-8).…”

“…This compound reverses the ataxic effects of ethanol (40) and blocks the effects of ethanol at δ-GABA A Rs (3,8). Initially there were hopes that Ro15-4513 would have a significant impact on the clinical treatment of alcohol-use disorders.…”

“…The δ-GABA A Rs also represent one of the major targets of ethanol in the CNS, particularly at relatively low ethanol concentrations, and mediate some of the rewarding and ataxic effects of ethanol (2)(3)(4)(5)(6)(7)(8). For instance, a genetic polymorphism that exaggerates the actions of ethanol at δ-GABA A Rs also potentiates ethanolinduced motor impairment (2).…”

Alcohol Modulation of Extra-synaptic Gamma-aminobutyric Acid Type A Receptors

“…This is especially important as the extra-synaptic GABAARs are thought to be most sensitive to ethanol at levels of social drinking, i.e., less than 30 mM (Sundstrom-Poromaa et al, 2002;Wallner et al, 2003Wallner et al, , 2006Wei et al, 2003;Hanchar et al, 2005;Santhakumar et. al., 2007;Glykys et al, 2007;Olsen et al, 2007).…”