Subunit Stoichiometry of Oligomeric Membrane Proteins: GABAA Receptors Isolated by Selective Immunoprecipitation from the Cell Surface
Section snippets
Monoclonal antibodies and cDNAs
bd17 recognizes epitopes on both β2 and β3 subunits, the reaction being stronger with β3 than with β2; bd24 is selective for the α1 subunit of human and bovine, but not rat, origin and recognizes an extracellular part of this subunit (Schoch et al., 1985; Ewert et al., 1990; Benke et al., 1991).
The human α1 and the rat β3 subunit of the GABAA receptor were used in this study. Human α1 is identical with the reported sequence (Schofield et al., 1989) except for the substitution Arg → Trp at
Characterization of α1β3 GABAA receptors expressed in Xenopus oocytes
Xenopus oocytes expressing the subunit combination α1β3 of the GABAA receptor were voltage-clamped and exposed to different concentrations of GABA. The expression of average maximal GABA current amplitudes > 6 μA indicates that this GABAA receptor subunit combination resulted in efficient formation of channels. A typical dose-response curve is shown in Fig. 2. The EC50 (apparent affinity for GABA) and Hill coefficient derived from a fit to the standardized average GABA dose-response curve are
DISCUSSION
We have investigated the subunit stoichiometry of GABAA receptors composed of α1 and β3 subunits expressed in Xenopus oocytes. Newly expressed GABAA receptors were characterized using electrophysiological, immunological methods and were in appropriate cases labeled with [35S]methionine. Isolation of mature receptors specifically from the surface membrane was followed by separation of the subunits. The ratio of α/β subunits obtained was 1.1 ± 0.1 (SEM, n = 6). Based on these data we can not
CONCLUSION
The ratio found here by using metabolic labeling in conjunction with immunoprecipitation directly from the plasma membrane predicts either a tetrameric GABAA receptor 2α2β, or favors 3α2β for a pentameric receptor. It excludes a 3:1 and a 1:3 subunit stoichiometry for a tetrameric receptor and a 4:1 and a 1:4 stoichiometry for a pentameric αβ receptor. The methods established here will help to identify the subunit stoichiometry of other GABAA receptor subunit combinations, or any other cloned
Acknowledgements
We are grateful to Prof. H. Reuter for continuous support. We thank V. Gyoerffy and V. Kiefer for skillful technical assistance. The support to E.S. of the Swiss National Science Foundation and the Foundation for the Promotion of Scientific Research at the University of Bern is gratefully acknowledged.
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Comparison of γ-aminobutyric acid, type A (GABA<inf>A</inf>), receptor αβγ and αβδ expression using flow cytometry and electrophysiology: Evidence for alternative subunit stoichiometries and arrangements
2016, Journal of Biological ChemistryCitation Excerpt :For αβδ receptors, support for an analogous stoichiometry of 2α:2β:1δ has been provided by atomic force microscopy (8), site-directed mutagenesis (9), and pharmacological studies (10), with a subunit arrangement of β-α-β-α-δ having been proposed in one study (8). However, alternative receptor stoichiometries and arrangements have been proposed in other studies, not only for αβδ receptors (11–13) but also for αβ (14, 15), αβγ (16–20), and αβϵ receptors (21). The lack of consensus regarding GABAA receptor subunit stoichiometry and arrangement likely reflects the highly variable methodologies employed to date.
Subunit Arrangement of γ-Aminobutyric Acid Type A Receptors
2001, Journal of Biological ChemistryCitation Excerpt :We can exclude tetrameric receptors of the subunit arrangements αβαβ from the expression of each of the tandem constructs alone and the arrangement αββα from their co-expression. Only arrangements of a 1:1 stoichiometry of α and β subunits have been tested here because stoichiometries for αβ receptors of 3:1 or 1:3 have been shown to be unlikely (19, 20). These findings confirm the conclusion drawn from Western blot analysis that proteolytic cleavage in the sequence of the linker (Fig.7A) does not occur to a significant extent.
Two invariant tryptophans on the α1 subunit define domains necessary for GABA(A) receptor assembly
1999, Journal of Biological ChemistryRecent advances in GABA research
1999, Neurochemistry InternationalStructure and subunit composition of GABA(A) receptors
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1999, Molecular and Cellular Neurosciences
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Present address: Department of Pharmacology, University of Washington, Seattle, WA 98195, U.S.A.