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: 9.81). 06/2006; 103(22):8540-5. DOI: 10.1073/pnas.0600194103
Source: PubMed

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.

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Available from: Martin Wallner, Aug 09, 2015
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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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    • "To determine whether this variability resulted from differences in the amount of ␦ subunit-incorporation, we exploited a " functional tag, " a mutation in the ␦ subunit (␦H68A) that conferred diazepam sensitivity to ␣4␤3␦H68A receptors with no changes in EtOH or GABA sensitivity. Using the ␦H68A mutation, we found that the magnitudes of EtOH, ␤-CCE [another allosteric modulator at the EtOH/ Ro15-4513 site in ␣4␤3␦ receptors (Hanchar et al., 2006; Wallner et al., 2006b)], and DZ enhancement covary in individual recordings. This is consistent with our hypothesis that incomplete ␦ subunit incorporation causes variability in EtOH responses in recombinant systems. "
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    ABSTRACT: GABA(A) receptors (GABA(A)Rs) have long been a focus as targets for alcohol actions. Recent work suggests that tonic GABAergic inhibition mediated by extrasynaptic δ subunit-containing GABA(A)Rs is uniquely sensitive to ethanol and enhanced at concentrations relevant for human alcohol consumption. Ethanol enhancement of recombinant α4β3δ receptors is blocked by the behavioral alcohol antagonist 8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester (Ro15-4513), suggesting that EtOH/Ro15-4513-sensitive receptors mediate important behavioral alcohol actions. Here we confirm alcohol/alcohol antagonist sensitivity of α4β3δ receptors using human clones expressed in a human cell line and test the hypothesis that discrepant findings concerning the high alcohol sensitivity of these receptors are due to difficulties incorporating δ subunits into functional receptors. To track δ subunit incorporation, we used a functional tag, a single amino acid change (H68A) in a benzodiazepine binding residue in which a histidine in the δ subunit is replaced by an alanine residue found at the homologous position in γ subunits. We demonstrate that the δH68A substitution confers diazepam sensitivity to otherwise diazepam-insensitive α4β3δ receptors. The extent of enhancement of α4β3δH68A receptors by 1 μM diazepam, 30 mM EtOH, and 1 μM β-carboline-3-carboxy ethyl ester (but not 1 μM Zn(2+) block) is correlated in individual recordings, suggesting that δ subunit incorporation into recombinant GABA(A)Rs varies from cell to cell and that this variation accounts for the variable pharmacological profile. These data are consistent with the notion that δ subunit-incorporation is often incomplete in recombinant systems yet is necessary for high ethanol sensitivity, one of the features of native δ subunit-containing GABA(A)Rs.
    Molecular pharmacology 11/2010; 78(5):918-24. DOI:10.1124/mol.109.062687 · 4.12 Impact Factor
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    • "Binding to the benzodiazepine site in GABA A Rs is known to cause both positive and negative (inverse) agonist modulation. Recent studies suggest that the benzodiazepine inverse agonist Ro15-4513 antagonizes alcohol by binding to a non-benzodiazepine binding site in the EC domain (Mehta and Ticku, 1989;Wallner et al., 2006). Moreover, hyperbaric studies suggest that pressure antagonizes ethanol-induced chloride uptake by uncoupling a benzodiazepinelike allosteric modulation of the receptor (Davies and Alkana, 2003). "
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    ABSTRACT: Glycine receptors (GlyRs) are recognized as the primary mediators of neuronal inhibition in the spinal cord, brain stem and higher brain regions known to be sensitive to ethanol. Building evidence supports the notion that ethanol acting on GlyRs causes at least a subset of its behavioral effects and may be involved in modulating ethanol intake. For over two decades, GlyRs have been studied at the molecular level as targets for ethanol action. Despite the advances in understanding the effects of ethanol in vivo and in vitro, the precise molecular sites and mechanisms of action for ethanol in ligand-gated ion channels in general, and in GlyRs specifically, are just now starting to become understood. The present review focuses on advances in our knowledge produced by using molecular biology, pressure antagonism, electrophysiology and molecular modeling strategies over the last two decades to probe, identify and model the initial molecular sites and mechanisms of ethanol action in GlyRs. The molecular targets on the GlyR are covered on a global perspective, which includes the intracellular, transmembrane and extracellular domains. The latter has received increasing attention in recent years. Recent molecular models of the sites of ethanol action in GlyRs and their implications to our understanding of possible mechanism of ethanol action and novel targets for drug development in GlyRs are discussed.
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