ArticleStudies of ethanol actions on recombinant δ-containing γ-aminobutyric acid type A receptors yield contradictory results
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).
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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 α4:β3:γ2S and 1:1:10 for α4:β3: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.
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