Elsevier

Neuropharmacology

Volume 43, Issue 4, September 2002, Pages 695-700
Neuropharmacology

Interactive report
The relative amount of cRNA coding for γ2 subunits affects stimulation by benzodiazepines in GABAA receptors expressed in Xenopus oocytes

https://doi.org/10.1016/S0028-3908(02)00036-9Get rights and content

Abstract

Benzodiazepine (BZD) potentiation of GABA-activated Cl-current (IGABA) in recombinant GABAA receptors requires the presence of the γ subunit. When α1, β2 and γ2S cRNA are expressed in a 1:1:1 ratio in Xenopus oocytes, BZD potentiation of IGABA is submaximal, variable and diminishes over time. Potentiation by BZDs is increased, more reproducible and is stabilized over time by increasing the relative amount of cRNA coding for the γ2S subunit. In addition, GABA EC50 values for α1β2γ2 (1:1:1) receptors are intermediate to values measured for α1β2 (1:1) and α1β2γ2 (1:1:10) receptors. We conclude that co-expression of equal ratios of α1, β2 and γ2 subunits in Xenopus oocytes produces a mixed population of α1β2 and α1β2γ2 receptors. Therefore, for accurate measurements of BZD potentiation it is necessary to inject a higher ratio of γ2 subunit cRNA relative to α1 and β2 cRNA. This results in a purer population of α1β2γ2 receptors.

Introduction

BZDs exert anxiolytic and anti-epileptic effects in the central nervous system by allosteric modulation of IGABA in GABAA receptors. Because of the widespread clinical utility of these drugs, understanding the mechanisms by which BZDs alter ion channel function is an important pharmacological inquiry. One action of this class of drugs is to increase inhibitory post-synaptic transmission, either by slowing desensitization or by enhancing chloride currents. BZDs cause a leftward shift in the GABAA receptor responsiveness to its neurotransmitter (Sigel and Baur, 1988, Zhang et al., 1993, Mellor and Randall, 1997). The structural properties of the BZD binding site have recently been reviewed (Sigel and Buhr, 1997).

GABAA receptors are heteromeric protein complexes composed of five subunits arranged pseudo-symmetrically around a central Cl-selective channel. The majority of the GABAA receptors in the brain are likely to be comprised of a combination of α1, β1, β2 and γ2 subunits (Benke et al., 1991, Laurie et al., 1992, Benke et al., 1994, Stephenson, 1995). For α1β2γ2 receptors, the most common stoichiometry is likely 2α:2β:1γ (Im et al., 1995, Chang et al., 1996, Tretter et al., 1997, Farrar et al., 1999). While co-expression of α1 and β2 subunits leads to formation of functional receptors, the γ2 subunit is required for high affinity BZD binding (Pritchett et al., 1989), and potentiation of IGABA by positive modulatory BZDs at subsaturating concentrations of GABA (Pritchett et al., 1989, Lavoie and Twyman, 1996, Perrais and Ropert, 1999). The extent of potentiation varies greatly depending on the report. Here, we present data that help explain this variability. In previous studies, we found that over-expressing either wild-type γ2 subunits or chimeric γ2-containing subunits with lower amounts of wild-type α1 and β2 subunits (1:1:10 α1:β2:γ2 or chimera) led to more consistent results in measures of BZD modulation of IGABA (Buhr et al., 1997, Buhr and Sigel, 1997, Boileau et al., 1998, Boileau and Czajkowski, 1999). It was also observed that BDZ potentiation was larger on average and more stable over time upon relative overexpression of the γ2 subunit. Here, we document these observations. Our data are restricted to observations in Xenopus oocytes and do not necessarily apply to mammalian cells although similar observations have been made in transfected HEK-293 cells (R. Furtmüller, personal communication; Czajkowski laboratory, data not shown).

Section snippets

Construction of receptor subunits

Data were obtained in the laboratories of C.C. and E.S., using different strategies for the construction of the receptor subunits. Both strategies are detailed below. The cDNAs coding for the α1, β2, and γ2S subunits of the rat GABAA receptor channel have been described elsewhere (Lolait et al., 1989, Malherbe et al., 1990a, Malherbe et al., 1990b).

In the E.S. laboratory, the cDNAs were subcloned into the polylinker of pBC/CMV. This expression vector allows high level expression under a SP6

Results

In two laboratories using different methods for oocyte and cRNA preparation, different cRNA vectors, and different solutions and techniques for two-electrode voltage-clamp recording, we find a requirement for over-expression of the γ2 subunit in α1β2γ2 receptors in order to attain consistent diazepam potentiation of IGABA. Fig. 1A shows that potentiation of IGABA by diazepam reaches a higher maximum value, with less variability (2.8 ± 0.2), for α1β2γ2 receptors expressed in a 1:1:10 rather than

Discussion

In the literature, a wide range of values has been given for the potentiation by BZD of currents elicited by GABA in recombinant α1β2γ2 GABAA receptors. In this report, possible causes for this variability are suggested. In most studies, a cRNA stoichiometry of α1:β2:γ2 = 1:1:1 has been used. An α1:β2:γ2 ratio of 1:1:1 may already be biased toward α1β2γ2 receptors, since they only require one γ for every two α and β subunits (Im et al., 1995, Chang et al., 1996, Tretter et al., 1997, Farrar et

Acknowledgements

This work was supported by a NINDS grant NS34727 to C.C and the Swiss National Science Foundation grants 3100-053599.98/1 and 3100-064789.01/1 to E.S.

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