Interactive reportThe relative amount of cRNA coding for γ2 subunits affects stimulation by benzodiazepines in GABAA receptors expressed in Xenopus oocytes
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.
References (41)
- et al.
Subunit arrangement of gamma-aminobutyric acid type A receptors
J. Biol. Chem.
(2001) - et al.
GABAA receptors display association of gamma 2-subunit with alpha 1- and beta 2/3-subunits
J. Biol. Chem.
(1991) - et al.
Distribution, prevalence, and drug binding profile of gamma-aminobutyric acid type A receptor subtypes differing in the beta-subunit variant
J. Biol. Chem.
(1994) - et al.
Subcellular localization and endocytosis of homomeric gamma2 subunit splice variants of gamma-aminobutyric acid type A receptors
Mol. Cell. Neurosci.
(1999) - et al.
Functional and molecular distinction between recombinant rat GABAA receptor subtypes by Zn2+
Neuron
(1990) - et al.
Stoichiometry of a ligand-gated ion channel determined by fluorescence energy transfer
J. Biol. Chem.
(1999) - et al.
Chloride channel expression with the tandem construct of alpha 6-beta 2 GABAA receptor subunit requires a monomeric subunit of alpha 6 or gamma 2
J. Biol. Chem.
(1995) - et al.
In vitro RNA synthesis with SP6 RNA polymerase
Methods Enzymol.
(1987) - et al.
Direct evidence for diazepam modulation of GABAA receptor microscopic affinity
Neuropharmacology
(1996) - et al.
Subunit stoichiometry of a mammalian K+ channel determined by construction of multimeric cDNAs
Neuron
(1992)
Cloning and expression of a novel rat GABAA receptor
FEBS Lett.
GABAA-receptor expressed from rat brain alpha- and beta-subunit cDNAs displays potentiation by benzodiazepine receptor ligands
Brain Res. Mol. Brain Res.
Sequence and functional characterization of a renal sodium/dicarboxylate cotransporter
J, Biol. Chem.
Potassium currents expressed from Drosophila and mouse eag cDNAs in Xenopus oocytes
Neuropharmacology
The benzodiazepine binding site of GABAA receptors
Trends Pharmacol. Sci.
The effect of subunit composition of rat brain GABAA receptors on channel function
Neuron
Synthesis of long, capped transcripts in vitro by SP6 and T7 RNA polymerases
Methods Enzymol.
GABA(A) receptor cell surface number and subunit stability are regulated by the ubiquitin-like protein Plic-1
Nat. Neurosci.
Identification of transduction elements for benzodiazepine modulation of the GABA(A) receptor: three residues are required for allosteric coupling
J. Neurosci.
Molecular dissection of benzodiazepine binding and allosteric coupling using chimeric gamma-aminobutyric acid type A receptor subunits
Mol. Pharmacol.
Cited by (105)
A neolignan from Connarus tuberosus as an allosteric GABA<inf>A</inf> receptor modulator at the neurosteroid binding site
2023, Biomedicine and PharmacotherapyDefined concatenated α6α1β3γ2 GABA<inf>A</inf> receptor constructs reveal dual action of pyrazoloquinolinone allosteric modulators
2019, Bioorganic and Medicinal ChemistryCitation Excerpt :Xenopus laevis oocytes were prepared, injected and defolliculated as described previously41,42. Oocytes were injected with 50 nL of the cRNA solution containing either wild type rat α1/α6β3γ2 subunits at a concentration of 10 :10 :50 nM for the building of loose receptors43, or the constructed dual and triple concatemers. The dual (β3γ2, β3α6) and triple (α6β3α6, α6β3α1, α1β3α1, α1β3α6, γ2β3α1) constructs were injected each at 50 nM either alone or in different combinations (at a ratio of 25 : 25 nM), resulting in a total of four different concatenated receptors.
An intersubunit electrostatic interaction in the GABA<inf>A</inf> receptor facilitates its responses to benzodiazepines
2018, Journal of Biological Chemistry