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

Department of Molecular and Medical Pharmacology, University of California, Los Angeles, CA 90095-1735, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 06/2006; 103(22):8540-5. DOI: 10.1073/pnas.0600194103
Source: PubMed


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.

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    • "Presumably, this Ro15-4513 sensitive tonic current that was ablated was normally composed of α4βγ2-containing GABA A -Rs since they are markedly potentiated by Ro15-4513 whereas α4δ-containing receptors are not (Wallner et al., 2006b). Even though α4δ receptors are not potentiated by Ro15-4513, they still bind this compound with high affinity (Hanchar et al., 2006) and it has been proposed that this binding to α4δ receptors antagonizes the effects of ethanol (Hanchar et al., 2006; Wallner et al., 2006b). However, our previously reported results that Ro15-4513 antagonism of ethanol-induced LORR was normal in δ subunit KO mice (Mihalek et al., 2001) argue against this. "
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    ABSTRACT: Alcohol (ethanol) is widely consumed for its desirable effects but unfortunately has strong addiction potential. Some imidazobenzodiazepines such as Ro15-4513 are able to antagonize many ethanol-induced behaviors. Controversial biochemical and pharmacological evidence suggest that the effects of these ethanol antagonists and ethanol are mediated specifically via overlapping binding sites on α4/δ-containing GABA(A)-Rs. To investigate the requirement of α4-containing GABA(A)-Rs in the mechanism of action of Ro15-4513 on behavior, wildtype (WT) and α4 knockout (KO) mice were compared for antagonism of ethanol-induced motor incoordination and hypnosis. Motor effects of ethanol were tested in two different fixed speed rotarod assays. In the first experiment, mice were injected with 2.0 g/kg ethanol followed 5 min later by 10 mg/kg Ro15-4513 (or vehicle) and tested on a rotarod at 8 rpm. In the second experiment, mice received a single injection of 1.5 g/kg ethanol ± 3 mg/kg Ro15-4513 and were tested on a rotarod at 12 rpm. In both experiments, the robust Ro15-4513 antagonism of ethanol-induced motor ataxia that was observed in WT mice was absent in KO mice. A loss of righting reflex (LORR) assay was used to test Ro15-4513 (20 mg/kg) antagonism of ethanol (3.5 g/kg)-induced hypnosis. An effect of sex was observed on the LORR assay, so males and females were analyzed separately. In male mice, Ro15-4513 markedly reduced ethanol-induced LORR in WT controls, but α4 KO mice were insensitive to this effect of Ro15-4513. In contrast, female KO mice did not differ from WT controls in the antagonistic effects of Ro15-4513 on ethanol-induced LORR. We conclude that Ro15-4513 requires α4-containing receptors for antagonism of ethanol-induced LORR (in males) and motor ataxia.
    Frontiers in Pharmacology 04/2011; 2:18. DOI:10.3389/fphar.2011.00018 · 3.80 Impact Factor
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    • "We suggest that these receptors that are sensitive to EtOH levels resulting from one glass of wine (3-30 mM) are certain subtypes of GABAA-R, the δ subunit-containing extrasynaptic GABAA-R that mediate tonic inhibition.15,16 One major target of anesthetic doses of EtOH (30-100 mM) is in the transmembrane domain of GABAA-Rs and glycine receptors.13,17,18 "
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    ABSTRACT: The purpose of this review is to summarize current knowledge of detailed biochemical evidence for the role of γ-aminobutyric acid type A receptors (GABA(A)-Rs) in the mechanisms of general anesthesia. With the knowledge that all general anesthetics positively modulate GABA(A)-R-mediated inhibitory transmission, site-directed mutagenesis comparing sequences of GABA(A)-R subunits of varying sensitivity led to identification of amino acid residues in the transmembrane domain that are critical for the drug actions in vitro. Using a photo incorporable analogue of the general anesthetic, R(+)etomidate, we identified two transmembrane amino acids that were affinity labelled in purified bovine brain GABA(A)-R. Homology protein structural modelling positions these two residues, αM1-11' and βM3-4', close to each other in a single type of intersubunit etomidate binding pocket at the β/α interface. This position would be appropriate for modulation of agonist channel gating. Overall, available information suggests that these two etomidate binding residues are allosterically coupled to sites of action of steroids, barbiturates, volatile agents, and propofol, but not alcohols. Residue α/βM2-15' is probably not a binding site but allosterically coupled to action of volatile agents, alcohols, and intravenous agents, and α/βM1-(-2') is coupled to action of intravenous agents. Establishment of a coherent and consistent structural model of the GABA(A)-R lends support to the conclusion that general anesthetics can modulate function by binding to appropriate domains on the protein. Genetic engineering of mice with mutation in some of these GABA(A)-R residues are insensitive to general anesthetics in vivo, suggesting that further analysis of these domains could lead to development of more potent and specific drugs.
    Canadian Anaesthetists? Society Journal 02/2011; 58(2):206-15. DOI:10.1007/s12630-010-9429-7 · 2.53 Impact Factor
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    • "Some reports suggested that Ro 15-4513 inhibits ethanol-induced potentiation of GABAA receptor function in brain membrane vesicles and cultured neurons (Suzdak et al., 1986; Mehta and Ticku, 1988), making this simple receptor antagonism the possible mode of action. More recently, ethanol has been suggested to competitively inhibit the binding of Ro 15-4513 to α4/6βδ subunit-containing GABAA receptors (Hanchar et al., 2006; Wallner et al., 2006). For some neuronal classes (cerebellar granule cells, thalamic relay neurons, dentate granule cells), these receptor subtypes make up a large portion of extrasynaptic receptors that are exceptionally sensitive to GABA, and which do not desensitize. "
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    ABSTRACT: Ethyl alcohol (ethanol) has many molecular targets in the nervous system, its potency at these sites being low compared to those of sedative drugs. This has made it difficult to discover ethanol's binding site(s). There are two putative binding sites at γ-aminobutyric acid (GABA) type A receptor subtypes for the proposed ethanol antagonist Ro 15-4513, the established γ2 subunit-dependent benzodiazepine site and the recently reported δ subunit-dependent Ro 15-4513/ethanol binding site. Here, we aimed at clarifying the in vivo role of Ro 15-4513 at these two sites. We found that the antagonism of ethanol actions by Ro 15-4513 in wildtype mice was dependent on the test: an open field test showed that light sedation induced by 1.5-1.8 g/kg ethanol was sensitive to Ro 15-4513, whereas several tests for ethanol-induced anxiolytic effects showed that the ethanol-induced effects were insensitive to Ro 15-4513. Antagonism of ethanol-induced sedation by Ro 15-4513 was unaffected in GABA(A) receptor δ subunit knockout mice. By contrast, when testing the GABA(A) receptor γ2 subunit F77I knock-in mouse line (γ2I77 mice) with its strongly reduced affinity of the benzodiazepine sites for Ro 15-4513, we found that the ethanol-induced sedation was no longer antagonized by Ro 15-4513. Indeed, γ2I77 mice had only a small proportion of high-affinity binding of [(3)H]Ro 15-4513 left as compared to wildtype mice, especially in the caudate-putamen and septal areas, but these residual sites are apparently not involved in ethanol antagonism. In conclusion, we found that Ro 15-4513 abolished the sedative effect of ethanol by an action on γ2 subunit-dependent benzodiazepine sites.
    Frontiers in Neuroscience 01/2011; 5(1):3. DOI:10.3389/fnins.2011.00003 · 3.66 Impact Factor
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