Elsevier

Alcohol

Volume 41, Issue 3, May 2007, Pages 155-162
Alcohol

Article
Studies of ethanol actions on recombinant δ-containing γ-aminobutyric acid type A receptors yield contradictory results

https://doi.org/10.1016/j.alcohol.2007.03.006Get rights and content

Abstract

The γ-aminobutyric acid type A receptors (GABAA-Rs) display a wide variety of subunit combinations. Drugs such as benzodiazepines have shown differential effects based on GABAA-R subunit composition. Actions of alcohols and volatile anesthetics generally do not vary markedly with subunit composition, with low concentrations of ethanol being poor modulators of these receptors. Recent studies showed α4/6- and δ-containing GABAA-Rs (located extrasynaptically and responsible for tonic currents in selective brain regions) presenting high sensitivity to low concentrations of ethanol, but these results have not been obtained in other laboratories. We carried out additional experiments varying the receptor level of expression, and GABA and ethanol concentration, but no sensitivity to low concentrations of ethanol was detected. We will discuss these results and attempt an analysis of the possible causes for the discrepancies.

Introduction

Gifted with a long list of subunits (6 α, 3 β, 3 γ, δ, ɛ, π, and θ, several of which possess isoforms), the pentameric γ-aminobutyric acid type A receptor (GABAA-R) has an incredible number of potential subunit combinations, at least in theory. In practice, a comparatively reduced number of combinations have been observed, with the most abundant being α1β2γ2S (Sieghart and Sperk, 2002, Whiting et al., 1999). Two combinations that have attracted attention lately are α4β2/3δ and α6β2/3δ: even though they are not very abundant, they present a more restricted expression, with respect to both regional and neuronal localization, and a very distinctive pharmacology. Numerous studies have provided evidence supporting the coexpression of α4 and δ subunits in brain, mainly in hippocampal dentate granule cells, thalamocortical relay neurons, and outer layers of the cerebral cortex (Korpi et al., 2002, Mihalek et al., 1999, Nusser et al., 1999, Peng et al., 2002, Pirker et al., 2000, Sur et al., 1999). In the cerebellum, δ is coexpressed with α6 subunits in the granule cells (Jechlinger et al., 1998, Jones et al., 1997, Pirker et al., 2000, Tretter et al., 2001). The localization of δ-containing GABAA-Rs is nonsynaptic (Nusser et al., 1998, Sun et al., 2004, Wei et al., 2003). Their higher GABA affinity makes them exquisitely sensitive to the low GABA concentrations in the perisynapsis and extrasynaptic space, which produces a small tonic current, in contrast with the phasic current observed in the synapsis, where GABAA-Rs contain γ subunits (Farrant and Nusser, 2005).

Section snippets

Expression in heterologous systems

While studies of brain slices or homogenates can render valuable information, the characterization of the properties of a particular subunit combination is usually better achieved by expressing the subunits of interest in heterologous systems, therefore controlling the subunit composition of the receptor. Attractive as the approach is, it is not without caveats: the host cell may influence the receptor properties, and it may be difficult to achieve a level of expression that allows the study of

Behavioral experiments with δ-knockout mice

The generation and subsequent testing of δ-knockout mice (Mihalek et al., 1999) revealed reduced effects of neuroactive steroids, specifically, decreased sedative, anxiolytic, and pro-absence seizure effects. The precise molecular basis for these differences is not clear yet. For instance, THDOC modulation of evoked and spontaneous inhibitory postsynaptic currents (IPSCs) in thalamocortical relay neurons was not modified in δ-knockout mice (Porcello et al., 2003), but both THDOC (Vicini et al.,

Effects of ethanol on α4/6- and δ-containing GABAA-Rs

The results obtained in brain slice preparations is covered in this issue by Valenzuela and others, and will not be reviewed in this article.

Further experiments in our laboratory

The methodology used was the same as that described in Borghese et al. (2006). Essentially, Xenopus laevis oocytes were isolated and injected with cRNAs encoding the GABAA subunits α4, β3, δ, and/or γ2S in the ratios 1:1:3 for α432S and 1:1:10 for α43:δ, unless otherwise indicated (the injected amounts of α4 and β3 were always 0.4 ng each per oocyte). Six to nine days after injection, recordings were carried out using the two-electrode voltage clamp technique.

Possible basis for the discrepancy

We tried several experimental approaches to identify the factors that control sensitivity to low concentrations of ethanol in α4β3δ GABAA-Rs, but none of them produced a different result. So far, there is no ready and easy explanation for the discrepancy in results between ten research groups, a situation not entirely new in the ethanol field.

Differences in system used [transient expression in oocytes and CHO cells, stable cell line L(tk)], clones (rat vs. human), and genetic backgrounds in

Acknowledgments

This study was supported by National Institutes of Health Grants AA06399 and GM47818, and by the Waggoner Center for Alcohol and Addiction Research.

References (48)

  • V. Tretter et al.

    Targeted disruption of the GABA(A) receptor delta subunit gene leads to an up-regulation of gamma 2 subunit-containing receptors in cerebellar granule cells

    J. Biol. Chem.

    (2001)
  • S. Vicini et al.

    GABA(A) receptor delta subunit deletion prevents neurosteroid modulation of inhibitory synaptic currents in cerebellar neurons

    Neuropharmacology

    (2002)
  • M. Wallner et al.

    Low dose acute alcohol effects on GABA(A) receptor subtypes

    Pharmacol. Ther.

    (2006)
  • C.M. Borghese et al.

    The delta subunit of gamma-aminobutyric acid type A receptors does not confer sensitivity to low concentrations of ethanol

    J. Pharmacol. Exp. Ther.

    (2006)
  • N. Brown et al.

    Pharmacological characterization of a novel cell line expressing human alpha(4)beta(3)delta GABA(A) receptors

    Br. J. Pharmacol.

    (2002)
  • P.A. Davies et al.

    Insensitivity to anaesthetic agents conferred by a class of GABA(A) receptor subunit

    Nature

    (1997)
  • M. Farrant et al.

    Variations on an inhibitory theme: phasic and tonic activation of GABA(A) receptors

    Nat. Rev. Neurosci.

    (2005)
  • J.L. Fisher et al.

    Single channel properties of recombinant GABAA receptors containing gamma 2 or delta subtypes expressed with alpha 1 and beta 3 subtypes in mouse L929 cells

    J. Physiol.

    (1997)
  • K.F. Haas et al.

    GABAA receptor subunit gamma2 and delta subtypes confer unique kinetic properties on recombinant GABAA receptor currents in mouse fibroblasts

    J. Physiol.

    (1999)
  • H.J. Hanchar et al.

    Ethanol potently and competitively inhibits binding of the alcohol antagonist Ro15-4513 to alpha4/6beta3delta GABAA receptors

    Proc. Natl. Acad. Sci. USA

    (2006)
  • A.M. Hosie et al.

    Zinc-mediated inhibition of GABA(A) receptors: discrete binding sites underlie subtype specificity

    Nat. Neurosci.

    (2003)
  • M. Jechlinger et al.

    Subunit composition and quantitative importance of hetero-oligomeric receptors: GABAA receptors containing alpha6 subunits

    J. Neurosci.

    (1998)
  • A. Jones et al.

    Ligand-gated ion channel subunit partnerships: GABAA receptor alpha6 subunit gene inactivation inhibits delta subunit expression

    J. Neurosci.

    (1997)
  • R.M. Mihalek et al.

    Attenuated sensitivity to neuroactive steroids in gamma-aminobutyrate type A receptor delta subunit knockout mice

    Proc. Natl. Acad. Sci. USA

    (1999)
  • Cited by (0)

    View full text