Subunit Stoichiometry of Oligomeric Membrane Proteins: GABAA Receptors Isolated by Selective Immunoprecipitation from the Cell Surface

doi:10.1016/S0028-3908(96)00034-2

Neuropharmacology

Volume 35, Issues 9–10, 1996, Pages 1403-1411

https://doi.org/10.1016/S0028-3908(96)00034-2 Get rights and content

Abstract

GABA_A receptors are hetero-oligomeric proteins of unknown subunit stoichiometry. In this study α1β3 GABA_A receptor channels were functionally expressed in Xenopus oocytes. Direct immunoprecipitation from the oocyte surface was used to exclusively isolate mature GABA_A receptors. The subunit ratio was determined by quantitation of the amount of [³⁵S]methionine incorporated into individual receptor subunits. Antibody released from the antigen or antibody not reacted was prevented from reassociation with labeled antigen by addition of excess unlabeled antigen. Variation of the α1β3 ratio of injected cRNAs only slightly affected the subunit ratio in mature receptors. This indicates that the subunit stoichiometry generated is independent of the pools of newly synthesized subunit monomers and supports the view that the receptor assembly is a regulated process. The ratio of $α1 β3$ subunits was found to be 1.1 ± 0.1 (SEM, n = 6). Our data are in best agreement with a tetrameric receptor with the composition 2α2β. For a pentameric receptor the ratio found slightly favors a receptor with the composition 3α2β. The method developed here is applicable to the determination of the subunit stoichiometry of other recombinant oligomeric membrane proteins. Copyright © 1996 Elsevier Science Ltd

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 GABA_A 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 GABA_A receptors expressed in Xenopus oocytes

Xenopus oocytes expressing the subunit combination α1β3 of the GABA_A receptor were voltage-clamped and exposed to different concentrations of GABA. The expression of average maximal GABA current amplitudes > 6 μA indicates that this GABA_A 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 GABA_A receptors composed of α1 and β3 subunits expressed in Xenopus oocytes. Newly expressed GABA_A 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 GABA_A 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 GABA_A 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
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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.
The subunit stoichiometry and arrangement of synaptic αβγ GABA_A receptors are generally accepted as 2α:2β:1γ with a β-α-γ-β-α counterclockwise configuration, respectively. Whether extrasynaptic αβδ receptors adopt the analogous β-α-δ-β-α subunit configuration remains controversial. Using flow cytometry, we evaluated expression levels of human recombinant γ2 and δ subunits when co-transfected with α1 and/or β2 subunits in HEK293T cells. Nearly identical patterns of γ2 and δ subunit expression were observed as follows: both required co-transfection with α1 and β2 subunits for maximal expression; both were incorporated into receptors primarily at the expense of β2 subunits; and both yielded similar FRET profiles when probed for subunit adjacency, suggesting similar underlying subunit arrangements. However, because of a slower rate of δ subunit degradation, 10-fold less δ subunit cDNA was required to recapitulate γ2 subunit expression patterns and to eliminate the functional signature of α1β2 receptors. Interestingly, titrating γ2 or δ subunit cDNA levels progressively altered GABA-evoked currents, revealing more than one kinetic profile for both αβγ and αβδ receptors. This raised the possibility of alternative receptor isoforms, a hypothesis confirmed using concatameric constructs for αβγ receptors. Taken together, our results suggest a limited cohort of alternative subunit arrangements in addition to canonical β-α-γ/δ-β-α receptors, including β-α-γ/δ-α-α receptors at lower levels of γ2/δ expression and β-α-γ/δ-α-γ/δ receptors at higher levels of expression. These findings provide important insight into the role of GABA_A receptor subunit under- or overexpression in disease states such as genetic epilepsies.
Subunit Arrangement of γ-Aminobutyric Acid Type A Receptors
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Citation 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.
The GABA_A receptors are ligand-gated chloride channels. The subunit stoichiometry of the receptors is controversial; four, five, or six subunits per receptor molecule have been proposed for αβ receptors, whereas αβγ receptors are assumed to be pentamers. In this study, α-β and β-α tandem cDNAs from the α1 and β2 subunits of the GABA_A receptor were constructed. We determined the minimal length of the linker that is required between the two subunits for functional channel expression for each of the tandem constructs. 10- and 23-amino acid residues are required for α-β and β-α, respectively. The tandem constructs either alone or in combination with each other failed to express functional channels in Xenopusoocytes. Therefore, we can exclude tetrameric or hexameric αβ GABA_A receptors. We can also exclude proteolysis of the tandem constructs. In addition, the tandem constructs were combined with single α, β, or γ subunits to allow formation of pentameric arrangements. In contrast to the combination with α subunits, the combination with either β or γ subunits led to expression of functional channels. Therefore, a pentameric arrangement containing two α1 and three β2 subunits is proposed for the receptor composed of α and β subunits. Our findings also favor an arrangement βαγβα for the receptor composed of α, β, and γ subunits.
Two invariant tryptophans on the α1 subunit define domains necessary for GABA(A) receptor assembly
1999, Journal of Biological Chemistry
Two invariant tryptophan residues on the N-terminal extracellular region of the rat α1 subunit, Trp-69 and Trp-94, are critical for the assembly of the GABA_A(γ-aminobutyric acid, type A) receptor into a pentamer. These tryptophans are common not only to all GABA_A receptor subunits, but also to all ligand-gated ion channel subunits. Converting each Trp residue to Phe and Gly by site-directed mutagenesis allowed us to study the role of these invariant tryptophan residues. Mutant α1 subunits, coexpressed with β2 subunits in baculovirus-infected Sf9 cells, displayed high affinity binding to [³H]muscimol, a GABA site ligand, but no binding to [³⁵S]t-butyl bicyclophosphorothionate, a ligand for the receptor-associated ion channel. Neither [³H]muscimol binding to intact cells nor immunostaining of nonpermeabilized cells gave evidence of surface expression of the receptor. When expressed with β2 and γ2 polypeptides, the mutant α1 polypeptides did not form [³H]flunitrazepam binding sites though wild-type α1 polypeptides did. The distribution of the mutant receptors on sucrose gradients suggests that the effects on ligand binding result from the inability of the mutant α1 subunits to form pentamers. We conclude that Trp-69 and Trp-94 participate in the formation of the interface between α and β subunits, but not of the GABA binding site.
Recent advances in GABA research
1999, Neurochemistry International
In this article I throw attention on to this GABA issue by outlining several aspects of current interest in the field of GABA research. The theme was selected in association with the Pharmacology and Therapeutical Potential of the GABA System symposium of the Second European Congress of Pharmacology held in July 1999 in Budapest, Hungary. A wide range of topics relating to the GABA system were outlined, including new members of the GABA_A receptor gene family, subunit composition of native GABA_A receptors, surface expression and clustering of GABA_A receptor subunits, allosteric modulation of GABA_A receptors, localization of agonist binding sites, GABA release, GABA_A–GABA_B receptor crosstalk, GABA_A and GABA_B receptor functions in different brain areas, altered transport and GABA_A receptor pattern in different models of epilepsy.
Structure and subunit composition of GABA(A) receptors
1999, Neurochemistry International
GABA_A receptors are the major inhibitory neurotransmitter receptors in the brain and are the site of action of many clinically important drugs. These receptors are composed of five subunits that can belong to eight different subunit classes. If all GABA_A receptor subunits could randomly combine with each other, an extremely large number of GABA_A receptor subtypes with distinct subunit composition and arrangement would be formed. Depending on their subunit composition, these receptors would exhibit distinct pharmacological and electrophysiological properties. Recent evidence, however, indicates that not all subunits can assemble efficiently with each other and form functional homo- or hetero-oligomeric receptors. In addition, the efficiency of formation of hetero-oligomeric assembly intermediates determines the subunit stoichiometry and subunit arrangement for each receptor and thus further reduces the possible heterogeneity of GABA_A receptors in the brain. Studies investigating the subunit composition of native GABA_A receptors support this conclusion, but also indicate that receptors composed of one, two, three, four, or five different subunits might exist in the brain. Using a recently established immunodepletion technique, the subunit composition and quantitative importance of native GABA_A receptor subtypes can be determined. This information, together with studies on the regional, cellular and subcellular distribution of these receptor subtypes, will be the basis for a rational development of drugs that specifically affect the GABAergic system.
Subcellular localization and endocytosis of homomeric γ2 subunit splice variants of γ-aminobutyric acid type A receptors
1999, Molecular and Cellular Neurosciences
The expression of α and β γ-aminobutyric acid type A receptor subunits produces GABA-gated channels which require the incorporation of either the γ2 or γ3 subunit for benzodiazepine modulation. Here we examine the role of the γ2 subunit splice variants, γ2S and γ2L which differ by eight amino acids in the major intracellular domain, in mediating cell surface expression. Using immunocytochemistry we have demonstrated that when expressed alone, the γ2S subunit can access the cell surface and internalize constitutively. In contrast, α1, β2 and γ2L are retained predominantly in the endoplasmic reticulum (ER) when expressed alone. Replacing the insert which differentiates γ2L from γ2S (LLRMFSFK) with eight alanines produces a phenotype identical to γ2S. Both γ2 subunits fail to produce high molecular weight oligomers observed for α1β2 and α1β2γ2 heterooligomers and do not form functional ion channels. Surface expression of γ2S is repressed upon the coexpression of α1 or β2 subunits, resulting in ER-retained heterooligomers, suggesting that homomeric γ2S is unlikely to occurin vivo. However, its independent maturation to surface competence and preferential assembly with α and β subunits may ensure the production of functional benzodiazepine-sensitive receptors. Furthermore, the presence of the γ2 subunit appears to confer an endocytotic capacity to these heterooligomeric receptors.

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^†: Present address: Department of Pharmacology, University of Washington, Seattle, WA 98195, U.S.A.

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Subunit Stoichiometry of Oligomeric Membrane Proteins: GABAA Receptors Isolated by Selective Immunoprecipitation from the Cell Surface

Abstract

Section snippets

Monoclonal antibodies and cDNAs

Characterization of α1β3 GABAA receptors expressed in Xenopus oocytes

DISCUSSION

CONCLUSION

Acknowledgements

J. biol. Chem.

Neuroscience

J. biol. Chem.

Int. Rev. Neurobiol.

Curr. Opin. struc. Biol.

TiNS

FEBS Lett.

Neuron

Neuron

FEBS Lett.

J. biol. Chem.

J. biol. Chem.

Neuron

J. biol. Chem.

Cell 72/Neuron (Suppl.)

Neuron

GABAA receptor needs two homologous domains of the β-subunit for activation by GABA but not by pentobarbital

Nature

Assembly of GABAA receptor subunits: α1β1 and α1β1γ2S subunits produce unique ion channels with dissimilar singlechannel conductance

J. Neurosci.

Assembly of GABAA receptor subunits: analysis of single-cell expression utilizing a fluorescent substrate/marker technique

J. Neurosci.

Stoichiometry of a recombinant GABAA receptor deduced from mutation-induced rectification

NeuroReport

Subunit Stoichiometry of Oligomeric Membrane Proteins: GABA_A Receptors Isolated by Selective Immunoprecipitation from the Cell Surface

Characterization of α1β3 GABA_A receptors expressed in Xenopus oocytes

GABA_A receptor needs two homologous domains of the β-subunit for activation by GABA but not by pentobarbital

Assembly of GABA_A receptor subunits: α₁β₁ and α₁β_1γ2S subunits produce unique ion channels with dissimilar singlechannel conductance

Assembly of GABA_A receptor subunits: analysis of single-cell expression utilizing a fluorescent substrate/marker technique

Stoichiometry of a recombinant GABA_A receptor deduced from mutation-induced rectification