Low-dose alcohol actions on alpha4beta3delta GABAA 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" alpha4/6beta3delta GABA(A) receptors at doses that do not reduce the GABA-induced Cl(-) current. At low ethanol concentrations (< or =30 mM), the Ro15-4513 antagonism is complete. However, at higher ethanol concentrations (> or =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 beta3 subunit (beta3N265M). Therefore, alpha4/6beta3delta GABA receptors have two distinct alcohol modulation sites: (i) a low-dose ethanol site present in alpha4/6beta3delta 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 alpha4beta3N265Mdelta 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 alpha4beta3delta 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.

Fig. 1.

Ro15-4513 antagonizes ethanol effects on recombinant α4β3δ receptor currents. (a and b) To mimic tonic GABA current, 300 nM GABA was perfused onto Xenopus oocytes expressing rat α4β3δ receptors that were held at −80 mV, and currents were measured with a two-electrode voltage clamp system. The indicated doses of ethanol and drugs were applied. (a) Ro15-4513 (300 nM) completely antagonized ethanol enhancement up to an ethanol concentration of 30 mM. (b) Cumulative Ro15-4513 dose-response curve shows a dose-dependent inhibition of ethanol effects. GABA-evoked currents were blocked by 30 μM bicuculline. (c) GABA peak currents with and without ethanol and the indicated concentrations of Ro15-4513. Ro15-4513 led to a dose-dependent inhibition of (10, 30, and 50 mM) ethanol enhancement on α4β3δ receptors. At concentrations up to 300 nM, Ro15-4513 did not block the basal current on α4β3δ receptors (evoked by 300 nM GABA). (a and b) Representative recordings of six and five experiments, respectively.

Fig. 2.

Ro15-4513 alcohol antagonism is antagonized by flumazenil and β-CCE, but not flunitrazepam and DMCM. (a) Currents were evoked by 300 nM GABA and potentiated by 30 mM ethanol, and this potentiation was reversed by 100 nM Ro15-4513. In constant presence of 300 nM GABA, 30 mM ethanol, and 100 nM Ro15-4513, the BZ site ligands Ro15-1788 (300 nM), β-CCE (300 nM), flunitrazepam, and DMCM (each 1 μM) were sequentially applied to test whether they reverse Ro15-4513’s ethanol antagonist effects. At the end of the recording, 30 μM bicuculline was used to block the GABA-induced current. Shown is a representative recording of a total of three experiments. (b) Chemical structures of the imidazobenzodiazepines Ro15-4513 and Ro15-1788 show that they differ only at one single position in the molecule. The clinically used BZ antagonist flumazenil (Ro15-1788) carries a fluorine at the C7 position of the BZ ring, whereas Ro15-4513 carries the larger azido group.

Fig. 3.

β-CCE enhances ethanol effects and is an agonist on α4β3δ receptors. (a) The β-carboline β-CCE at the indicated concentrations was applied alone or together with ethanol (always in the presence of 300 nM GABA) to oocytes expressing α4β3δ receptors, and peak GABA/Cl⁻ currents were measured. (b) Dose-dependent reversal of 300 nM β-CCE enhancement of α4β3δ currents by Ro15-4513.

Fig. 4.

A point mutation eliminates Ro15-4513-insensitive alcohol effects at high alcohol concentrations. (a) A single point mutation (β3N265M in membrane-spanning segment TM2 of the β3 subunit) abolishes the Ro15-4513-resistant alcohol enhancement observed at high ethanol concentrations in α4β3N265Mδ receptors. (b) Alcohol dose-response curve, determined by brief coapplications of ethanol and GABA EC₂₀ (300 nM for α4β3δ and α4β3N265Mδ, and 30 μM GABA for α4β3γ2 GABA_AR). Currents through α4β3N265Mδ GABA_AR show a saturable alcohol enhancement and an EC₅₀ of around 16 mM. The complete reversal of even very high dose alcohol effects by 300 nM Ro15-4513 in these experiments is surprising, because this behavior is not expected from an ideal competitive relationship of ligands with apparent dissociation constants (10 nM for Ro15-4513 and 16 mM for ethanol). The reasons why Ro15-4513 is so potent in antagonizing high-dose ethanol actions on functional receptors remain to be clarified.