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Forced Subunit Assembly in α1β2γ2 GABAAReceptors

2002; Elsevier BV; Volume: 277; Issue: 48 Linguagem: Inglês

10.1074/jbc.m207663200

1083-351X

Sabine W. Baumann, Roland Baur, Erwin Sigel,

Ion channel regulation and function

The major isoform of the γ-aminobutyric acid type A (GABAA) receptor is thought to be composed of 2α1, 2β2, and 1γ2 subunit(s), which surround the ion pore. Definite evidence for the subunit arrangement is lacking. We show here that GABAA receptor subunits can be concatenated to a trimer that can be functionally expressed upon combination with a dimer. Many combinations did not result in the functional expression. In contrast, four different combinations of triple subunits with dual subunit constructs, all resulting in the identical pentameric receptor γ2β2α1β2α1, could be successfully expressed in Xenopus oocytes. We characterized the functional properties of these receptors in respect to agonist, competitive antagonist, and diazepam sensitivity. All properties were similar to those of wild type α1β2γ2 GABAAreceptors. Thus, together with information on the crystal structure of the homologous acetylcholine-binding protein (Brejc, K., van Dijk, W. J., Klaassen, R. V., Schuurmans, M., van Der Oost, J., Smit, A. B., and Sixma, T. K., (2001) Nature411, 269–276, we provide evidence for an arrangement γ2β2α1β2α1, counterclockwise when viewed from the synaptic cleft. Forced subunit assembly will also allow receptors containing different subunit isoforms or mutant subunits to be expressed, each in a desired position. The methods established here should be applicable to the entire ion channel family comprising nicotinic acetylcholine, glycine, and 5HT3 receptors. The major isoform of the γ-aminobutyric acid type A (GABAA) receptor is thought to be composed of 2α1, 2β2, and 1γ2 subunit(s), which surround the ion pore. Definite evidence for the subunit arrangement is lacking. We show here that GABAA receptor subunits can be concatenated to a trimer that can be functionally expressed upon combination with a dimer. Many combinations did not result in the functional expression. In contrast, four different combinations of triple subunits with dual subunit constructs, all resulting in the identical pentameric receptor γ2β2α1β2α1, could be successfully expressed in Xenopus oocytes. We characterized the functional properties of these receptors in respect to agonist, competitive antagonist, and diazepam sensitivity. All properties were similar to those of wild type α1β2γ2 GABAAreceptors. Thus, together with information on the crystal structure of the homologous acetylcholine-binding protein (Brejc, K., van Dijk, W. J., Klaassen, R. V., Schuurmans, M., van Der Oost, J., Smit, A. B., and Sixma, T. K., (2001) Nature411, 269–276, we provide evidence for an arrangement γ2β2α1β2α1, counterclockwise when viewed from the synaptic cleft. Forced subunit assembly will also allow receptors containing different subunit isoforms or mutant subunits to be expressed, each in a desired position. The methods established here should be applicable to the entire ion channel family comprising nicotinic acetylcholine, glycine, and 5HT3 receptors. The γ-aminobutyric acid type A (GABAA) 1The abbreviations used for: GABAA, γ-aminobutyric acid type A; DZ, diazepam. 1The abbreviations used for: GABAA, γ-aminobutyric acid type A; DZ, diazepam.receptors are the major inhibitory neurotransmitter receptors in the mammalian brain. They are heteromeric protein complexes consisting of five subunits, which are arranged pseudo-symmetrically around a central Cl−-selective channel (1Macdonald R.L. Olsen R.W. Annu. Rev. Neurosci. 1994; 17: 569-602Crossref PubMed Scopus (1768) Google Scholar). 18 different subunit isoforms have been cloned so far (1Macdonald R.L. Olsen R.W. Annu. Rev. Neurosci. 1994; 17: 569-602Crossref PubMed Scopus (1768) Google Scholar, 2Rabow L.E. Russek S.J. Farb D.H. Synapse. 1995; 21: 189-274Crossref PubMed Scopus (457) Google Scholar, 3Davies P.A. Hanna M.C. Hales T.G. Kirkness E.F. Nature. 1997; 385: 820-823Crossref PubMed Scopus (368) Google Scholar, 4Whiting P.J. McAllister G. Vassilatis D. Bonnert T.P. Heavens R.P. Smith D.W. Hewson L. O'Donnell R. Rigby M.R. Sirinathsinghji D.J. Marshall G. Thompson S.A. Wafford K.A. Vasilatis D. J. Neurosci. 1997; 17: 5027-5037Crossref PubMed Google Scholar, 5Hedblom E. Kirkness E.F. J. Biol. Chem. 1997; 272: 15346-15350Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 6Whiting P.J. Bonnert T.P. McKernan R.M. Farrar S. Le Bourdelles B. Heavens R.P. Smith D.W. Hewson L. Rigby M.R. Sirinathsinghji D.J. Thompson S.A. Wafford K.A. Ann. N. Y. Acad. Sci. 1999; 868: 645-653Crossref PubMed Scopus (297) Google Scholar, 7Barnard E.A. Skolnick P. Olsen R.W. Möhler H. Sieghart W. Biggio G. Braestrup C. Bateson A.N. Langer S.Z. Pharmacol. Rev. 1998; 50: 291-313PubMed Google Scholar). The major receptor isoform of the GABAA receptor in the brain most probably consists of α1, β2, and γ2 subunits (1Macdonald R.L. Olsen R.W. Annu. Rev. Neurosci. 1994; 17: 569-602Crossref PubMed Scopus (1768) Google Scholar,2Rabow L.E. Russek S.J. Farb D.H. Synapse. 1995; 21: 189-274Crossref PubMed Scopus (457) Google Scholar, 8Laurie D.J. Seeburg P.H. Wisden W. J. Neurosci. 1992; 12: 1063-1076Crossref PubMed Google Scholar, 9Benke D. Fritschy J.M. Trzeciak A. Bannwarth W. Möhler H. J. Biol. Chem. 1994; 269: 27100-27107Abstract Full Text PDF PubMed Google Scholar, 10McKernan R.M. Whiting P.J. Trends Neurosci. 1996; 19: 139-143Abstract Full Text PDF PubMed Scopus (1069) Google Scholar). The γ subunit has been shown to be required for functional modulation of the receptor channels by benzodiazepines (11Pritchett D.B. Sontheimer H. Shivers B.D. Ymer S. Kettenmann H. Schofield P.R. Seeburg P.H. Nature. 1989; 338: 582-585Crossref PubMed Scopus (1143) Google Scholar, 12Sigel E. Baur R. Trube G. Möhler H. Malherbe P. Neuron. 1990; 5: 703-711Abstract Full Text PDF PubMed Scopus (515) Google Scholar). Different approaches have indicated a 2α:2β:1γ subunit stoichiometry for this receptor (13Backus K.H. Arigoni M. Drescher U. Scheurer L. Malherbe P. Möhler H. Benson J.A. Neuroreport. 1993; 5: 285-288Crossref PubMed Scopus (118) Google Scholar, 14Chang Y. Wang R. Barot S. Weiss D.S. J. Neurosci. 1996; 16: 5415-5424Crossref PubMed Google Scholar, 15Tretter V. Ehya N. Fuchs K. Sieghart W. J. Neurosci. 1997; 17: 2728-2737Crossref PubMed Google Scholar, 16Farrar S.J. Whiting P.J. Bonnert T.P. McKernan R.M. J. Biol. Chem. 1999; 274: 10100-10104Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). The inferred arrangement of subunits around the channel pore is hypothetical, based on the findings that the GABA-binding site is located at intersubunit contacts between α and β subunits (17Sigel E. Baur R. Kellenberger S. Malherbe P. EMBO J. 1992; 11: 2017-2023Crossref PubMed Scopus (170) Google Scholar, 18Amin J. Weiss D.S. Nature. 1993; 366: 565-569Crossref PubMed Scopus (372) Google Scholar, 19Smith G.B. Olsen R.W. J. Biol. Chem. 1994; 269: 20380-20387Abstract Full Text PDF PubMed Google Scholar, 20Westh-Hansen S.E. Rasmussen P.B. Hastrup S. Nabekura J. Noguchi K. Akaike N. Witt M.R. Nielsen M. Eur. J. Pharmacol. 1997; 329: 253-257Crossref PubMed Scopus (43) Google Scholar, 21Boileau A.J. Evers A.R. Davis A.F. Czajkowski C. J. Neurosci. 1999; 19: 4847-4854Crossref PubMed Google Scholar) and that homologous amino acid residues of α and γ subunits form the benzodiazepine-binding pocket (22Wieland H.A. Luddens H. Seeburg P.H. J. Biol. Chem. 1992; 267: 1426-1429Abstract Full Text PDF PubMed Google Scholar, 23Amin J. Brooks-Kayal A. Weiss D.S. Mol. Pharmacol. 1997; 51: 833-841Crossref PubMed Scopus (106) Google Scholar, 24Buhr A. Sigel E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8824-8829Crossref PubMed Scopus (92) Google Scholar, 25Buhr A. Baur R. Sigel E. J. Biol. Chem. 1997; 272: 11799-11804Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 26Buhr A. Schaerer M.T. Baur R. Sigel E. Mol. Pharmacol. 1997; 52: 676-682Crossref PubMed Scopus (88) Google Scholar, 27Sigel E. Buhr A. Trends Pharmacol. Sci. 1997; 18: 425-429Abstract Full Text PDF PubMed Scopus (345) Google Scholar, 28Teissere J.A. Czajkowski C. J. Neurosci. 2001; 21: 4977-4986Crossref PubMed Google Scholar). The observation that assembly intermediates comprising αγ or αβ dimers displayed some benzodiazepine or agonist binding, respectively (29Klausberger T. Ehya N. Fuchs K. Fuchs T. Ebert V. Sarto I. Sieghart W. J. Biol. Chem. 2001; 276: 16024-16032Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), supported conclusions drawn from the former mutation studies. From the crystal structure of the acetylcholine-binding protein (30Brejc K. van Dijk W.J. Klaassen R.V. Schuurmans M. van Der Oost J. Smit A.B. Sixma T.K. Nature. 2001; 411: 269-276Crossref PubMed Scopus (1564) Google Scholar), a protein homologous to the extracellular domain of the nicotinic acetylcholine receptors and the other members of the superfamily of ligand-gated ion channels, we can deduce the absolute position of the amino acid residues involved in the formation of agonist and drug-binding sites in a subunit in the pentamer. However, this acetylcholine-binding protein is a homopentamer and gives no information about the arrangement of the heteromeric GABAA receptors. In a former study (31Baumann S.W. Baur R. Sigel E. J. Biol. Chem. 2001; 276: 36275-36280Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar) we expressed α-β and β-α tandem constructs in combination with single γ subunits inXenopus oocytes and investigated the function of the formed receptors. The results suggested a possible arrangement γβαβα; however, some uncertainty remained. From this work it also was not clear whether exclusively one or several arrangements of a given set of subunits is possible. In the present study we aimed to express GABAA receptors from various combinations of linked constructs containing two and three subunits. A single pentameric arrangement made of α1, β2, and γ2 subunits was found to result in functional ion channels. We describe its properties toward the agonist GABA, the competitive antagonist bicuculline, and the positive allosteric modulator diazepam. The described techniques will allow forced assembly and functional study of GABAA and related receptors of defined subunit arrangements. For simplicity, we use the following: α for modified rat α1, β for rat β2, and γ for rat γ2. The modified rat α subunit differs from the rat α by one amino acid residue, which confers the subunit-specific bd24 antibody recognition (32Ewert M. Shivers B.D. Lüddens H. Möhler H. Seeburg P.H. J. Cell Biol. 1990; 110: 2043-2048Crossref PubMed Scopus (137) Google Scholar, 33Häring P. Stähli C. Schoch P. Takacs B. Staehelin T. Möhler H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4837-4841Crossref PubMed Scopus (157) Google Scholar). This property has previously been used to exclude proteolysis of the linked constructs (31Baumann S.W. Baur R. Sigel E. J. Biol. Chem. 2001; 276: 36275-36280Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). The antibody only reacts if the N-terminal of the α subunit is free. Therefore, the same test was not feasible for most of the present constructs. Other evidence was used to make proteolysis unlikely (see below). Tandem constructs for γ-β, β-γ, γ-α, and α-γ with various linker lengths were made similar as described in Baumannet al. (31Baumann S.W. Baur R. Sigel E. J. Biol. Chem. 2001; 276: 36275-36280Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). In the following, numbers between two subunit symbols describe the length of the introduced synthetic linker. Triple constructs were prepared from tandem constructs as exemplified for the γ-26-β-23-α (γ-β-α) construct. The γ-26-β (γ-β) tandem construct was cut by HindIII in the β subunit and the vector behind the gene to yield a 7-kb fragment containing the sequence of the vector, the γ2 subunit, the linker, and the beginning of the β2 subunit. This vector fragment was dephosphorylated with shrimp alkaline phosphatase (USB) in 10 mm Tris-HCl, pH 8.0, and 100 mmMgCl2 for 1 h at 37 °C. The β-23-α (β-α) (31Baumann S.W. Baur R. Sigel E. J. Biol. Chem. 2001; 276: 36275-36280Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar) tandem construct was cut by HindIII in the β subunit and the vector behind the gene to yield a 2-kb fragment containing the sequence of the second half of the β subunit, the linker, and the α subunit. The two fragments were ligated, and proper ligation was checked by restriction analysis. The construct α-10-β-23-α (α-β-α) was made accordingly from α-10-β (31Baumann S.W. Baur R. Sigel E. J. Biol. Chem. 2001; 276: 36275-36280Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar) and β-23-α. The triple constructs β-23-α-10-γ (β-α-γ, from β-23-α and α-10-γ), γ-23-α-10-β (γ-α-β, from γ-23-α and α-10-β), and β-23-α-10-β (β-α-β from β-23-α and α-10-β) were prepared similarly using BamHI. The following linkers have been introduced: γ-26-β: Q5A3PAQ2(QA)2A2PA2Q5, α-10-γ: Q10, γ-23-α: Q3(Q2A3PA)2AQ5. Capped cRNAs were synthesized (Ambion, Austin, TX) from the linearized pCMV vectors containing the different tandem or triple constructs or the single α1, β2, and γ2subunits, respectively, and from the vector pVA2580 (34Kuhn F.J. Greeff N.G. J. Gen. Physiol. 1999; 114: 167-183Crossref PubMed Scopus (82) Google Scholar) encoding a neuronal voltage-gated sodium channel (Na). A poly-A tail of about 400 residues was added to each transcript using yeast poly-A polymerase (USB, Cleveland, OH). The concentration of the cRNA was quantified on a formaldehyde gel using Radiant Red stain (Bio-Rad) for visualization of the RNA and known concentrations of RNA ladder (Invitrogen) as standard on the same gel. cRNA combinations of triple/Na, triple/α/Na, triple/β/Na, triple/α-β/Na, triple/β-α/Na (triple = γ-β-α, γ-α-β, or β-α-γ), β-α/γ2/Na, β-α-β/α-γ/Na, and α-β-α/γ-β/Na were precipitated in ethanol/isoamylalcohol (19:1) and stored at −20 °C. For injection, the alcohol was removed and the cRNAs were dissolved in water. Isolation of oocytes from the frogs, culturing of the oocytes, injection of cRNA, and defolliculation were done as described earlier (35Sigel E. J. Physiol. 1987; 386: 73-90Crossref PubMed Scopus (113) Google Scholar). Oocytes were injected with 50 nl of the cRNA solution. For cRNA combinations of the γ2-containing triple constructs with the tandem construct, ratios of 50:10 nm and 10:10 nm were investigated. The combination of single α1, β2, and γ2 subunits was expressed at 10:10:50 nm. To allow standardization of expressed GABA currents cRNA coding for the voltage-gated sodium channel was always added to a concentration of 40 nm. The injected oocytes were incubated in modified Barth's solution (10 mm HEPES, pH 7.5, 88 mmNaCl, 1 mm KCl, 2.4 mm NaHCO3, 0.82 mm MgSO4, 0.34 mmCa(NO3)2, 0.41 mmCaCl2, 100 units/ml penicillin, 100 μg/ml streptomycin) at 18 °C for 2 days before the measurements. All measurements were done in medium containing 90 mm NaCl, 1 mmMgCl2, 1 mm KCl, 1 mmCaCl2, and 5 mm HEPES, pH 7.4, at a holding potential of −80 mV. For the determination of maximal current amplitudes 1 mm GABA (Fluka, Buchs, Switzerland) was applied for 20 s. Voltage-dependent sodium currents were determined by a potential jump from a holding potential of −100 to −15 mV. As the modes of activation of the GABA receptor channel and the voltage-dependent sodium channel differ, the measurements of the two channels do not interfere with each other. The GABA-evoked peak current amplitude was standardized to the co-expressed sodium current amplitude of the same oocyte. The mean standardized current amplitude of at least 3 oocytes per subunit combination was then compared with the mean standardized wild type current amplitude. GABA-evoked currents (at 8–12% of the maximal current amplitude) were inhibited by varying concentrations of bicuculline methiodide (RBI). Relative current stimulation by diazepam was determined at a GABA concentration evoking 2–5% of the maximal current amplitude in combination with varying concentrations of diazepam (DZ) (Roche) and expressed as ((I(GABA+DZ)/I(GABA))−1)×100%. We have shown previously (31Baumann S.W. Baur R. Sigel E. J. Biol. Chem. 2001; 276: 36275-36280Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar) that it is feasible to covalently link α and β subunits of the GABAA receptor while retaining full receptor function (31Baumann S.W. Baur R. Sigel E. J. Biol. Chem. 2001; 276: 36275-36280Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). These linked constructs allowed us to propose a possible arrangement of subunits in the α1β2γ2 receptor. However, some uncertainty remained due to a possible rearrangement of dual subunit constructs. We now have linked three subunits in different sequence and expressed them in combination with tandem constructs. Triple subunit constructs unlike dual subunit constructs cannot rearrange for topological reasons. In the following, the sequence of subunits in multiple subunit constructs is always described as the C-terminal of the first subunit linked to the N-terminal of the second subunit. To link the γ subunit to either side of the existing tandem constructs α-10-β and β-23-α, linker lengths for the new connections had to be established. To test functionality of these linkers, dual constructs α-γ, γ-α, β-γ, or γ-β were co-expressed with α-β or β-α constructs and single α or β subunits to yield receptors of the composition 2α2β1γ, e.g.γ-β/α-β/α (data not shown). The required linker lengths for functional expression were found to be 26 amino acid residues for γ-β, 10 amino acid residues for α-γ, and 23 residues for γ-α. For a β-γ tandem construct, linkers up to 25 amino acid residues in length were tested but were found to result only in very small functional channel expression. In the following, the triple constructs γ-β-α, β-α-γ, γ-α-β, β-α-β, and α-β-α were prepared. If the presence of 2α, 2β, and 1γ subunit(s) in a pentamer is assumed, six different arrangements of α, β, and γ subunits are possible (Fig. 1). Of this stoichiometry we tested first arrangements containing one γ and two each of α and β in an alternating fashion. Assembly studies of Tretter et al. (15Tretter V. Ehya N. Fuchs K. Sieghart W. J. Neurosci. 1997; 17: 2728-2737Crossref PubMed Google Scholar) suggest such an alternating arrangement because expression of either α and γ or β and γ leads to dimers only. In these cases either a β or α subunit is missing, respectively, to continue assembly. Expression of α and β, in contrast, leads to the formation of tetra- and pentamers. Two alternating arrangements are possible, namely γβαβα and γαβαβ. They are the most probable arrangements as they seem to contain the inferred two αβ and αγ subunit interfaces required for the formation of two GABA- and one benzodiazepine-binding sites. As the subunits are not symmetrical, only one of these two alternating arrangements is predicted to form the correct subunit interfaces for establishing binding sites. With our triple construct γ-α-β we show that the arrangement γαβαβ is not functional (Fig.2, second column). This was not unexpected after the earlier finding, which showed that expression of the α-β construct with single γ subunits resulted in decreased current amplitudes as compared with wild type receptors. γ-β-α did not result in functional channels upon expression in combination with γ, α-β, or γ-α, or very tiny currents in combination with α or β (Fig. 2, columns 4–8). In contrast, γ-β-α could be complemented with β-α (Fig.3). Similarly, β-α-γ cannot be complemented by α, β, or α-β (Fig. 2, columns 9–11) but by β-α (Fig. 3).Figure 3Maximal current amplitudes evoked by 1 mm GABA for several combinations of triple constructs with tandem constructs shown to result in the functional expression.The GABA-evoked current amplitudes were standardized to the current amplitude of the voltage-gated sodium channel expressed in the same oocyte. Measurements were in each case done in 5–6 oocytes from two different batches of oocytes.View Large Image Figure ViewerDownload (PPT) These observations all pointed to a channel with the subunit arrangement γβαβα. Fig. 3 shows indeed the successful functional expression of channels from different combinations of triple and dual subunit constructs. All these combinations (γ-β-α/β-α, β-α-γ/β-α, β-α-β/α-γ, and α-β-α/γ-β) actually result in the identical arrangement of subunits around the pore, and they can be seen as permutations of the positions of the linkers around the five subunits (Fig.4). When β-α-γ was expressed in combination with β-α at a stoichiometry of 1:1, currents amounted only to about 25% (10 oocytes, 2 batches of oocytes) of the amplitude observed upon expression of loose subunits α, β, and γ at 1:1:5. Normal expression levels were observed for β-α-γ and β-α expressed at 5:1 (Fig. 3). The reason for this increased requirement for β-α-γ is not clear. As mentioned before, receptors with a different stoichiometry and/or arrangement from the one discussed above were also tested for functional expression. Triple constructs γ-α-β and β-α-γ and dual constructs α-β and β-α (31Baumann S.W. Baur R. Sigel E. J. Biol. Chem. 2001; 276: 36275-36280Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar) when expressed alone did not result in functional channel expression. From the measurements of maximal current amplitudes evoked by application of GABA (Fig. 2) we can also exclude the arrangements γβααα, γβαββ, γβααβ, γααβα, γβββα, γαββα, γαβγγ, γβαγγ, and γβαγα. These results make it very likely that γβαβα is indeed the functional arrangement of the α1β2γ2receptor. The fact that no or very small currents were observed during linker length optimization and for many of the above mentioned subunit combinations indicates that proteolysis in the linker regions is not occurring to a significant extent. Small currents as observed, for example, for γ-β-α/α and γ-β-α/β can be thought to reflect mis-assembled channels. These channels are not necessarily silent, but are not formed to a significant extent. To characterize receptors with the subunit arrangement γβαβα made from linked constructs we investigated their response properties to the agonist GABA and the competitive antagonist bicuculline. First, we studied the GABA concentration-response properties of the tandem construct β-α in combination with single γ2 subunits. Compared with receptors made from single subunits (wild type) we observed a slight rightward shift (2.4-fold; Fig. 5 B). A similar 2-fold shift had already been observed for the combination of β-α with single β subunits (31Baumann S.W. Baur R. Sigel E. J. Biol. Chem. 2001; 276: 36275-36280Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Next, three of four combinations of triple constructs with tandem constructs that were proven functional before (Fig. 3) were characterized in their GABA concentration-response properties. Fig. 5 A shows representative current traces obtained with the subunit combination. All of them behaved very similarly, showing properties similar to wild type receptors regarding EC50 values (Fig. 5 and TableI). β-α-β/α-γ was only analyzed twice and had an average EC50 of 124 μm in these experiments.Table IAgonist, competitive antagonist, and benzodiazepine modulatory properties of identically arranged γβαβα receptors but made from different combinations of triple and dual subunit constructs. The data were averaged from 4–5 oocytes from 2 different batches of oocytes each. Data are given as mean ± SE.Subunit combinationEC50(GABA)Hill-coefficientIC50(Bicuculline)EC50 (Diazepam)μmμmnmα/β/γ51 ± 151.40 ± 0.180.76 ± 0.0492 ± 6β-α/γ123 ± 171.22 ± 0.081.16 ± 0.0938 ± 6γ-β-α/β-α177 ± 101.04 ± 0.021.65 ± 0.1982 ± 7β-α-γ/β-α101 ± 91.36 ± 0.051.33 ± 0.1662 ± 10α-β-α/γ-β118 ± 41.24 ± 0.071.10 ± 0.0551 ± 5 Open table in a new tab Fig. 6 shows a summary of inhibition experiments by the competitive inhibitor bicuculline of GABA-induced currents on all subunit construct combinations resulting in functional γβαβα receptors except β-α-β/α-γ. The obtained bicuculline concentration-response curves were very similar to those of the receptor made from single subunits. Table I summarizes the agonist and competitive antagonist properties of the different constructs. From these results we can conclude that the linkers used here between the subunits have little influence on the apparent affinity for GABA induced channel opening and the inhibition by bicuculline. It has earlier been observed that stimulation by diazepam of currents elicited by GABA in oocytes expressing α:β:γ = 1:1:1 is smaller and more variable than in oocytes expressing α:β:γ = 1:1:5 (36Boileau A.J. Baur R. Sharkey L.M. Sigel E. Czajkowski C. Neuropharmacology. 2002; 43: 695-700Crossref PubMed Scopus (101) Google Scholar). Therefore, it was interesting to examine receptors formed from triple and tandem constructs in this respect. The response to increasing concentrations of diazepam did not markedly differ from receptors containing loose subunits (Fig.7) regarding the concentration-dependence of current stimulation. However, there was a difference regarding maximal stimulation and variability in different oocytes of this value. Although stimulation by diazepam of currents elicited by GABA in oocytes expressing α:β:γ = 1:1:5 centered at about 170% and was quite variable in different oocytes, a value of about 270% with little variation was observed, provided the γ2 subunit was covalently linked to other subunits to give γ-β-α/β-α. Fig. 8 documents this by comparing diazepam stimulation in oocytes either expressing α:β:γ = 1:1:5 or covalently linked constructs γ-β-α:β-α = 1:1. The combinations β-α-γ/β-α, α-β-α/γ-β, and β-α/γ also showed values for maximal stimulation, which were higher than those found for wild type receptors and had small variations (data not shown). It has been observed earlier that the stimulation by diazepam decreases in oocytes injected with cRNA coding for α, β, and γ during expression time (36Boileau A.J. Baur R. Sharkey L.M. Sigel E. Czajkowski C. Neuropharmacology. 2002; 43: 695-700Crossref PubMed Scopus (101) Google Scholar). This phenomenon seemed not to occur in oocytes expressing linked subunits (not shown).Figure 8Comparison in extent and variability of the relative current stimulation by diazepam in α/β/γ GABAAreceptors and covalently linked γ-β-α/β-α receptors. The figure shows a frequency distribution. Data were grouped in bins for their stimulation by diazepam ((IGABA+DZ/IGABA)−1)×100%. The bin size was 40%. Frequency distributions were fitted with a Gauss distribution. Open bars are observations in non-linked receptors and closed bars in linked receptors.View Large Image Figure ViewerDownload (PPT) In the present study we used covalently linked subunits of the GABAA receptor to study the arrangement of subunits in α1β2γ2 receptors. We examined receptors made from combinations of triple and dual subunit constructs containing α1, β2, and/or γ2subunits in different orders. We present direct evidence that α1β2γ2 receptors have a subunit composition and relative arrangement of γβαβα from N-terminal to C-terminal. It should be stated that we restricted ourselves to looking at the ability of the subunit combinations to form functional ion channels. For combinations that did not result in function, we cannot say at present whether this is due to insufficient stability of constructs, or due to assembly problems, or whether the receptors reach the surface membrane and are unable to open. The majority of GABAA receptors contains α, β, and γ subunits (9Benke D. Fritschy J.M. Trzeciak A. Bannwarth W. Möhler H. J. Biol. Chem. 1994; 269: 27100-27107Abstract Full Text PDF PubMed Google Scholar, 37Fritschy J.M. Benke D. Mertens S. Oertel W.H. Bachi T. Mohler H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6726-6730Crossref PubMed Scopus (219) Google Scholar, 38Caruncho H.J. Costa E. Receptors Channels. 1994; 2: 143-153PubMed Google Scholar, 39Fritschy J.M. Mohler H. J. Comp. Neurol. 1995; 359: 154-194Crossref PubMed Scopus (1040) Google Scholar, 40Somogyi P. Fritschy J.M. Benke D. Roberts J.D. Sieghart W. Neuropharmacology. 1996; 35: 1425-1444Crossref PubMed Scopus (146) Google Scholar). This subunit composition can be reached by combining either three subunits of one type with one subunit each of the other two types or two subunits each from two types with one subunit of the third type. The former stoichiometry has been excluded for α3β2γ2 and α1β2γ2 receptors by electrophysiological characterization of mutant receptors (13Backus K.H. Arigoni M. Drescher U. Scheurer L. Malherbe P. Möhler H. Benson J.A. Neuroreport. 1993; 5: 285-288Crossref PubMed Scopus (118) Google Scholar, 14Chang Y. Wang R. Barot S. Weiss D.S. J. Neurosci. 1996; 16: 5415-5424Crossref PubMed Google Scholar). For the latter stoichiometry both receptors with 2α2β1γ and 2α1β2γ have been suggested. A 2α2β1γ stoichiometry is favored by electrophysiological data (14Chang Y. Wang R. Barot S. Weiss D.S. J. Neurosci. 1996; 16: 5415-5424Crossref PubMed Google Scholar), investigations using immunoprecipitation (15Tretter V. Ehya N. Fuchs K. Sieghart W. J. Neurosci. 1997; 17: 2728-2737Crossref PubMed Google Scholar), and fluorescence energy transfer studies (16Farrar S.J. Whiting P.J. Bonnert T.P. McKernan R.M. J. Biol. Chem. 1999; 274: 10100-10104Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). However, it has been reported that also two γ subunits can occur within the same pentamer. This possibility has been suggested on the basis of co-occurrence of isoforms of the γ subunit within the same receptor molecule, where γ2γ3 and γ2Sγ2L pairings have been detected in immunoprecipitation experiments (41Khan Z.U. Gutierrez A. De Blas A.L. J. Neurochem. 1996; 66: 685-691Crossref PubMed Scopus (80) Google Scholar, 42Quirk K. Gillard N.P. Ragan C.I. Whiting P.J. McKernan R.M. Mol. Pharmacol. 1994; 45: 1061-1070PubMed Google Scholar). We found that only combinations of triple and dual constructs, which resulted in the arrangement γβαβα, therefore containing 2α, 2β, and 1γ subunit(s), were functionally expressed with properties very similar to receptors made from single subunits. Combinations of subunits and/or linked subunit constructs that would result in a different stoichiometry or a different arrangement were not functional. Our study is limited to α1, β2, and γ2containing receptors. It is not clear whether receptors containing different isoforms of these subunit types have the same stoichiometry and arrangement as the α1β2γ2receptor. Taking into account that for the formation of GABA-binding sites defined interfaces must be formed, it is likely that all αβγ, αβδ, and αβε (4Whiting P.J. McAllister G. Vassilatis D. Bonnert T.P. Heavens R.P. Smith D.W. Hewson L. O'Donnell R. Rigby M.R. Sirinathsinghji D.J. Marshall G. Thompson S.A. Wafford K.A. Vasilatis D. J. Neurosci. 1997; 17: 5027-5037Crossref PubMed Google Scholar, 43Wisden W. Seeburg P.H. Curr. Opin. Neurobiol. 1992; 2: 263-269Crossref PubMed Scopus (231) Google Scholar) receptors follow the same building plan. When we expressed triple constructs in combination with tandem constructs to form γβαβα receptors we observed concentration-response properties of the resulting receptors very similar to receptors made from single, individual subunits. The linkers between the subunits do not strongly affect function. Only in one case did we observe a slight decrease in the Hill coefficient. This coefficient is 1.0 in the case of γ-β-α/β-α as compared with 1.2–1.4 for all other receptors (Table I). This observation might indicate a slightly altered gating behavior of the receptor channel. Horenstein et al. (44Horenstein J. Wagner D.A. Czajkowski C. Akabas M.H. Nat. Neurosci. 2001; 4: 477-485Crossref PubMed Scopus (136) Google Scholar) suggested an asymmetric turning of the subunits upon channel opening. They investigated αβ receptors and proposed that 4 subunits (2α and 2β) turn in the same direction, whereas 1β subunit turns in the opposite direction. This β subunit can be thought to be replaced by the γ subunit in the αβγ receptor. In this case the linker between γ and β in the γ-β-α construct could impair the suggested movement of the linked subunits, which could lead to the observed decrease of the Hill coefficient. This movement, however, did not seem to be disturbed in the γ-β dual construct when expressed in combination with the α-β-α triple construct (not shown). A dual subunit construct might be more flexible than a triple construct. Introduction of a shorter linker into the triple construct could confirm the above hypothesis. For all receptors formed from linked subunits we observed a 2.0–3.5-fold shift to the right in the GABA concentration-response curves compared with receptors made from single subunits. On one hand this shift could be due to changed binding or gating properties of the receptor pentamers by linkage. On the other hand it has been observed frequently that expression of single α, β, and γ subunits in oocytes or HEK cells leads to a mixed population of αβγ and αβ receptors (36Boileau A.J. Baur R. Sharkey L.M. Sigel E. Czajkowski C. Neuropharmacology. 2002; 43: 695-700Crossref PubMed Scopus (101) Google Scholar). The EC50 of αβ receptors is about 8 μm, that of αβγ receptors about 41 μm (45Sigel E. Baur R. J. Neurochem. 2000; 74: 2590-2596Crossref PubMed Scopus (38) Google Scholar). Expression of linked constructs does not allow the formation of pentameric αβ receptors, and the 2.0–3.5-fold shift of the concentration-response curve further to the right could at least be partly due to expression of pure αβγ receptors. This view is supported by the markedly higher stimulation of GABA-induced currents by diazepam of receptors made from linked subunits. Presence of αβ in αβγ receptors decreases apparent diazepam stimulation (36Boileau A.J. Baur R. Sharkey L.M. Sigel E. Czajkowski C. Neuropharmacology. 2002; 43: 695-700Crossref PubMed Scopus (101) Google Scholar). Our approach leads to the relative sequence of subunits in the receptor only, and does not allow a statement about the absolute arrangement,e.g. the sequence when viewed from the synaptic cleft. The determination of the crystal structure of the acetylcholine-binding protein (30Brejc K. van Dijk W.J. Klaassen R.V. Schuurmans M. van Der Oost J. Smit A.B. Sixma T.K. Nature. 2001; 411: 269-276Crossref PubMed Scopus (1564) Google Scholar) leads to an insight into the absolute configuration of the extracellular parts of the subunits and interfaces of the nicotinic acetylcholine receptor. The acetylcholine-binding protein is a bacterial protein homologous to the extracellular domain of the nicotinic acetylcholine receptor of higher organisms. The GABAA receptor belongs to the same superfamily of ligand-gated ion channels and is therefore structurally homologous to the nicotinic acetylcholine receptor. The knowledge of amino acid residues that form agonist and benzodiazepine-binding sites, respectively (for review see Ref. 27Sigel E. Buhr A. Trends Pharmacol. Sci. 1997; 18: 425-429Abstract Full Text PDF PubMed Scopus (345) Google Scholar) allows us to conclude on which side of the α subunit γ and β subunits are positioned when viewed from the synaptic cleft. Therefore, the absolute arrangement of the α1β2γ2 receptor is very likely γβαβα from the N terminus to the C terminus, read in anti-clockwise direction when viewed from the synaptic cleft (Fig. 9). This work will allow the study of the roles of any individual site located on an α or β subunit, which was impossible thus far because there were always two sites on the two subunits affected by a mutation in 2α2β1γ GABAA receptors. The possibility of a forced subunit assembly will enable targeted introduction of a mutation in only one subunit. This will, for example, allow dissection of the two low affinity agonist sites located at the αβ subunit interface. Forced subunit assembly will have further impact on the characterization of receptor forms containing different isoforms of the same subunit subtypes. Receptors that are made of 4 or 5 different subunits cannot be analyzed by recombinant expression of the mixture of the single subunits because many different receptor subtypes can be formed. Expression of predefined sequences of subunits in triple and tandem constructs will allow the study of pharmacological properties of defined receptor isoforms. As the GABAA receptor belongs to a superfamily of ligand-gated ion channels, methods described here are applicable to neuronal and non-neuronal nicotinic acetylcholine receptors, glycine receptors, and 5HT3 receptors. We thank Heleen van Hees for help in preparing the linked subunit constructs and Dr. V. Niggli for carefully reading the manuscript.