Subunit Arrangement of γ-Aminobutyric Acid Type A Receptors

Abstract

The GABAA 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 GABAA 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 GABAA 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. The GABAA 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 GABAA 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 GABAA 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. γ-aminobutyric acid GABA type A human embryonic kidney apparent affinity GABAA1receptors mediate fast synaptic inhibition in the mammalian brain. They are believed to form heterooligomers composed of subunits from six classes with several isoforms (α1–6, β1–3, γ1–3, ε, δ, θ, π) (1Macdonald R.L. Olsen R.W. Annu. Rev. Neurosci. 1994; 17: 569-602Crossref PubMed Scopus (1793) Google Scholar, 2Rabow L.E. Russek S.J. Farb D.H. Synapse. 1995; 21: 189-274Crossref PubMed Scopus (458) 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 (196) Google Scholar). These subunits belong to the gene superfamily of ligand-gated ion channels, which includes nicotinic acetylcholine receptors, GABAA receptors, glycine receptors, and the serotonin type 3 (5HT3) receptor. The major GABAA receptor isoform is likely to be composed of α1, β2, and γ2 subunits (1Macdonald R.L. Olsen R.W. Annu. Rev. Neurosci. 1994; 17: 569-602Crossref PubMed Scopus (1793) Google Scholar, 2Rabow L.E. Russek S.J. Farb D.H. Synapse. 1995; 21: 189-274Crossref PubMed Scopus (458) Google Scholar, 6Laurie D.J. Seeburg P.H. Wisden W. J. Neurosci. 1992; 12: 1063-1076Crossref PubMed Google Scholar, 7Benke D. Fritschy J.M. Trzeciak A. Bannwarth W. Möhler H. J. Biol. Chem. 1994; 269: 27100-27107Abstract Full Text PDF PubMed Google Scholar). Heterologous expression demonstrated that the combination of α and β subunits produces GABA-gated currents, but coexpression of a γ subunit is required for benzodiazepine sensitivity of the expressed receptors (8Pritchett D.B. Sontheimer H. Shivers B.D. Ymer S. Kettenmann H. Schofield P.R. Seeburg P.H. Nature. 1989; 338: 582-585Crossref PubMed Scopus (1149) Google Scholar). GABAA receptors composed of α and β subunits differ from receptors that additionally contain the γ subunit in regard to Zn2+ and benzodiazepine sensitivity and to single channel conductance (9Draguhn A. Verdoorn T.A. Ewert M. Seeburg P.H. Sakmann B. Neuron. 1990; 5: 781-788Abstract Full Text PDF PubMed Scopus (353) Google Scholar, 10Verdoorn T.A. Draguhn A. Ymer S. Seeburg P.H. Sakmann B. Neuron. 1990; 4: 919-928Abstract Full Text PDF PubMed Scopus (461) Google Scholar, 11Moss S.J. Ravindran A. Mei L. Wang J.B. Kofuji P. Huganir R.L. Burt D.R. Neurosci. Lett. 1991; 123: 265-268Crossref PubMed Scopus (41) Google Scholar, 12Smart T.G. Moss S.J. Xie X. Huganir R.L. Br. J. Pharmacol. 1991; 103: 1837-1839Crossref PubMed Scopus (209) Google Scholar, 13Angelotti T.P. Uhler M.D. Macdonald R.L. J. Neurosci. 1993; 13: 1418-1428Crossref PubMed Google Scholar). Some populations of neuronal GABAAreceptors show high Zn2+ sensitivity coupled with low single-channel conductance as described for αβ receptors (14Smart T.G. J. Physiol. 1992; 447: 587-625Crossref PubMed Scopus (120) Google Scholar, 15Kaneda M. Farrant M. Cull-Candy S.G. J. Physiol. 1995; 485: 419-435Crossref PubMed Scopus (197) Google Scholar). Although receptors made from α, β, and γ subunits are thought to be pentameric (16Backus 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, 17Chang Y. Wang R. Barot S. Weiss D.S. J. Neurosci. 1996; 16: 5415-5424Crossref PubMed Google Scholar, 18Tretter V. Ehya N. Fuchs K. Sieghart W. J. Neurosci. 1997; 17: 2728-2737Crossref PubMed Google Scholar), the subunit stoichiometry of receptors composed of α and β is still controversial. Recombinantly expressed receptors have been reported as possibly tetrameric (19Kellenberger S. Eckenstein S. Baur R. Malherbe P. Buhr A. Sigel E. Neuropharmacology. 1996; 35: 1403-1411Crossref PubMed Scopus (19) Google Scholar, 20Gorrie G.H. Vallis Y. Stephenson A. Whitfield J. Browning B. Smart T.G. Moss S.J. J. Neurosci. 1997; 17: 6587-6596Crossref PubMed Google Scholar) as well as pentameric (18Tretter V. Ehya N. Fuchs K. Sieghart W. J. Neurosci. 1997; 17: 2728-2737Crossref PubMed Google Scholar, 21Knight A.R. Hartnett C. Marks C. Brown M. Gallager D. Tallman J. Ramabhadran T.V. Receptor Channel. 1998; 6: 1-18PubMed Google Scholar). Unitary dose-response curves for αβ receptors, single IC50 values for Zn2+ inhibition, and unitary single channel properties (1Macdonald R.L. Olsen R.W. Annu. Rev. Neurosci. 1994; 17: 569-602Crossref PubMed Scopus (1793) Google Scholar) provide evidence against the formation of two populations of receptors, e.g. 2α3β and 3α2β. A tetrameric rather than a pentameric structure has been proposed as one of several explanations for the lower average single channel conductance for the αβ receptor as compared with the αβγ receptor (22Angelotti T.P. Macdonald R.L. J. Neurosci. 1993; 13: 1429-1440Crossref PubMed Google Scholar, 23Hevers W. Lüddens H. Mol. Neurobiol. 1998; 18: 35-86Crossref PubMed Scopus (412) Google Scholar). A powerful way to gain insight into the arrangement of subunits in a multimeric channel is to predetermine the alignment of subunits by gene fusion and to analyze whether the linked subunits are able to form functional channels. This approach was first successfully applied to potassium channels (24Isacoff E.Y. Jan Y.N. Jan L.Y. Nature. 1990; 345: 530-534Crossref PubMed Scopus (379) Google Scholar, 25Liman E.R. Tytgat J. Hess P. Neuron. 1992; 9: 861-871Abstract Full Text PDF PubMed Scopus (979) Google Scholar, 26Hurst R.S. Kavanaugh M.P. Yakel J. Adelman J.P. North R.A. J. Biol. Chem. 1992; 267: 23742-23745Abstract Full Text PDF PubMed Google Scholar). Later it was also used to study subunit stoichiometry of other ion channels, e.g. a cyclic nucleotide-gated channel (27Varnum M.D. Zagotta W.N. Biophys. J. 1996; 70: 2667-2679Abstract Full Text PDF PubMed Scopus (70) Google Scholar), the mechanosensitive channel MscL ofEscherichia coli (28Sukharev S.I. Schroeder M.J. McCaslin D.R. J. Membr. Biol. 1999; 171: 183-193Crossref PubMed Scopus (44) Google Scholar), and the cystic fibrosis conductance regulator channel (29Zerhusen B. Zhao J. Xie J. Davis P.B. Ma J. J. Biol. Chem. 1999; 274: 7627-7630Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). All these channels have their N and C termini on the cytoplasmic side so that the linkage occurs intracellularly. Up to now it has only been used once with limited success in the field of ligand-gated ion channels, which have both C and N termini on the extracellular side. Applying it to a GABAA receptor, Imet al. (30Im W.B. Pregenzer J.F. Binder J.A. Dillon G.H. Alberts G.L. J. Biol. Chem. 1995; 270: 26063-26066Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) prepared a tandem construct where the α6 subunit is linked to the β2 subunit via 10 glutamine residues and studied functional expression in HEK293 cells. The connection between the two subunits included the signal sequence of the β2 subunit of 24-amino acid residues in length. The consequences of such a signal sequence in the middle of a protein are difficult to predict. We constructed here tandem constructs of α1 and β2 subunits for the first time in both arrangements α1-β2 and β2-α1. We determined the minimal length of the linkers necessary for the formation of functional channels. The constructs were expressed in Xenopus either alone or in combination with single subunits to subunit stoichiometry and arrangement of We provide on the of GABAA receptors. α-β tandem cDNAs a single αβ with linkers of length were in the The tandem constructs of the α1 subunit M. Shivers B.D. Lüddens H. Möhler H. Seeburg P.H. J. Biol. 1990; PubMed Scopus Google Scholar) signal sequence the N and the β2 subunit the C The α1 subunit from the subunit by of one acid of this to the P. C. P. B. Möhler H. U. S. A. PubMed Scopus Google Scholar, M. Shivers B.D. Lüddens H. Möhler H. Seeburg P.H. J. Biol. 1990; PubMed Scopus Google Scholar), which was for in the The α1 subunit was by the as and the as a as a The was to the the and the first of the sequence for the linker The β2 subunit was as and the as as a The was to the first of the sequence of the protein and the of the sequence for the linker The the of the gene and sequence or The were in the sequence by or to be in a into the with and The sequence of the was In the the acid of the α1 subunit is to the first acid of the β2 In the other the acid are between the α1 subunit and the β2 The β-α tandem cDNAs were prepared The β subunit was as and as The α subunit was as and as The linkers into the β-α are the A sequence of glutamine residues the protein and to an of the Therefore, other acid residues were and residues were for their properties to form structure were from the containing the tandem the single α1, β2, and γ2 subunits, and from the J. Physiol. 1999; PubMed Scopus Google Scholar) a neuronal channel A of residues was to each S. The of the was on a for of the and of as the on the and and were in and the was and the were in of from the of the of and were as described E. J. Physiol. PubMed Scopus Google Scholar). were with of the of α1 and β2 subunits the each subunit or tandem construct In the of coexpression of α1, β2, and γ2 subunits, the α1 and β2 subunits or the tandem construct 10 and the γ2 subunit The channel was to a of The were in for the All were in containing and a of the of GABA was applied for were determined by a from a of to The was to the of the The of three per subunit combination was compared with the type by was determined a GABA of the in combination with were in 10 and each a The was on for and for The was to Nature. PubMed Scopus Google Scholar). were to to al. H. J. U. S. A. PubMed Scopus Google Scholar) and with the M. Shivers B.D. Lüddens H. Möhler H. Seeburg P.H. J. Biol. 1990; PubMed Scopus Google Scholar, P. C. P. B. Möhler H. U. S. A. PubMed Scopus Google Scholar), which the N of the α1 subunit of the GABAA receptor. were the cDNAs were constructed that of the α1 and the β2 subunit of the GABAA receptor in both α1-β2 and The α1 subunit was in form to into the by the signal The β2 subunit was of signal sequence it is difficult to consequences this of acid residues in the middle of the fusion protein the between the C of the α1 subunit and the N of the β2 we linkers of length to the linker in a functional fusion protein expression in the Xenopus Although the subunits of the GABAA receptor the and have a high sequence differ in the of acid residues the the C as well as the of the of the Therefore, linkers of length were for the α1-β2 and construct The of the tandem constructs was expression in Xenopus either alone and in combination with each other or in combination with single α1 and or β2 and subunits. of tandem constructs alone is to receptors composed of an of subunits, whereas combination of tandem constructs with single subunits can additionally in receptors with an of subunits. The first for channel was a with that of receptors made from single α1 and β2 subunits, the type receptor. These to of either α1 or β2 subunits alone failed to for in the expression between the were to the of the channel in the were for and the apparent affinity for GABA receptors to the type receptor were whether the expressed tandem constructs were or to proteolysis we the GABAA receptors by The against the of the α1 subunit M. Shivers B.D. Lüddens H. Möhler H. Seeburg P.H. J. Biol. 1990; PubMed Scopus Google Scholar, P. C. P. B. Möhler H. U. S. A. PubMed Scopus Google Scholar) was 3 that single α1 subunits of type receptors This is in the combination the of α1 subunit of proteolysis of the A signal the is also for by a the tandem construct The of with also proteolysis in the A containing that is than have been The tandem construct be the for the to the N of the α1 which is by the linker in this The of channel expressed with to described we also have functional evidence for the that proteolysis of both tandem constructs can be the α1-β2 tandem construct was with single β2 subunits, functional channels were the linker was linker but only the acid residues the of the α1 subunit to the N of the β2 was in a linker of residues in length we that expressed from type receptors, whereas the tandem construct with a linker of 10 residues in The dose-response curves of the and the tandem constructs were to that of the type receptors The two constructs in channels with of 3 and with the combination of single α and β subunits with of to an apparent affinity for GABA the curves of tandem constructs with single β2 the α-β tandem constructs with linkers of or acid residues in length in channels with an apparent affinity for channels from the β-α tandem construct with a linker of acid residues show a apparent affinity for whereas for channels from the construct with a linker of acid the apparent affinity is to the one of the combination of single α and β the of the of the for the constructs are was we combined the constructs with linkers of 10- and acid residues with single β2 subunits. A tandem construct with a linker of residues receptors with to of type receptors. the dose-response was to the the apparent affinity for GABA was was than that of the type receptors. A tandem construct containing a linker of residues also to type receptors. The GABA dose-response for these channels is by a of which is to the type receptor, with of The two functional tandem constructs and were either the or the constructs were expressed we GABA The channel the expression in tandem constructs or type receptors. the of and the of protein expression in the was we exclude proteolysis for either construct proteolysis of the linker in each α1 and β2 subunits, which in in functional channels. and constructs were expressed in the the of the type These led to the that tetrameric receptors of the arrangement which is to the arrangement or of the arrangement to a functional receptor made from single α and β subunits. The tandem constructs and were also with single α1 subunits and for was of GABA whereas were expressed in the The of a single α1 subunit to the combination of both tandem constructs and in as compared with the combination of with but were still of the type receptors. This an formation of functional channels in this that both the and the tandem constructs be with single β2 subunits to form functional channels. This the that both tandem constructs the arrangement with a single β2 subunit and the tandem construct is with a single γ2 the to compared with the type receptor can be by by The tandem construct with a single γ subunit in functional channels with to type receptors of these receptors are also by by In this study we have demonstrated the of subunit linkage α1-β2 and for the We have also the minimal linker required for functional Our a pentameric structure of the GABAA receptor composed of α1 and β2 subunits and exclude a The described here also be applied to the study of other ligand-gated ion channels. linkage of subunits is a powerful to stoichiometry and arrangement of multimeric This approach has first been applied to the study of potassium channels (24Isacoff E.Y. Jan Y.N. Jan L.Y. Nature. 1990; 345: 530-534Crossref PubMed Scopus (379) Google Scholar). al. (30Im W.B. Pregenzer J.F. Binder J.A. Dillon G.H. Alberts G.L. J. Biol. Chem. 1995; 270: 26063-26066Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) made a tandem construct of the GABAA receptor subunit α6 and They that their tandem construct alone failed to functional GABA channels, but combination with either single α6 or γ2 subunits, but β2 subunits, receptor expression in HEK293 cells. expression low in these and for the type subunit combination α6 and β2 (30Im W.B. Pregenzer J.F. Binder J.A. Dillon G.H. Alberts G.L. J. Biol. Chem. 1995; 270: 26063-26066Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). In the tandem constructs we the signal sequence of the which have protein of the protein into the subunit or proteolysis of the connection between the subunits. We linked the α1 and the β2 subunits of the GABAA receptor in both arrangements and expressed the tandem constructs α-β and β-α They were both to in functional channels with β2 subunits. the tandem constructs were expressed either alone or in combination with each functional receptors were Therefore, here is that the GABAA receptor made from α1 and β2 subunits is composed of an of subunits. We can exclude tetrameric receptors of the subunit arrangements from the expression of each of the tandem constructs alone and the arrangement from their arrangements of a stoichiometry of α and β subunits have been here for αβ receptors of or have been to be (19Kellenberger S. Eckenstein S. Baur R. Malherbe P. Buhr A. Sigel E. Neuropharmacology. 1996; 35: 1403-1411Crossref PubMed Scopus (19) Google Scholar, 20Gorrie G.H. Vallis Y. Stephenson A. Whitfield J. Browning B. Smart T.G. Moss S.J. J. Neurosci. 1997; 17: 6587-6596Crossref PubMed Google Scholar). These findings the from that in the sequence of the linker to a The of only one subunit of the tandem construct in the functional receptor can also be either one or both of these the formation of functional pentameric receptors from the tandem constructs alone have been The that both tandem constructs form functional channels in combination with single β2 subunits but to so in combination with single α1 subunits the that a receptor made from α and β subunits is a composed of two α and three β subunits. This also been proposed on in HEK293 receptors (18Tretter V. Ehya N. Fuchs K. Sieghart W. J. Neurosci. 1997; 17: 2728-2737Crossref PubMed Google Scholar). A receptor stoichiometry of three α6 and two β2 subunits has also been (30Im W.B. Pregenzer J.F. Binder J.A. Dillon G.H. Alberts G.L. J. Biol. Chem. 1995; 270: 26063-26066Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). This that the subunit stoichiometry of an αβ receptor on the subunit isoforms expressed on in the expression A of this study was the of linkers between the subunits. The linkage of two subunits both to each other in the receptor. functional channels can be the of the two subunits either channel or the linker is in functional channels can be expressed from linked subunits, their be assumed the linker is In this the two linked subunits to each other in the receptor is for subunit to between the two linked subunits. We determined the minimal linker length for the α-β and the β-α tandem necessary for the formation of functional channels. We this length to be 10 and acid respectively. linkers the apparent affinity for GABA or the of the by the of the receptor. be that the and tandem which have linkers that are 3 acid residues or as well as type receptors. Therefore, the linker length be than 10 or acid respectively. In of the linker length we included the linker as well as the and of the subunits We assumed an of both with per acid In this the length of the subunit connection be to be and in the α-β and the β-α tandem which be by the of structure the we that the linker length is is of to whether these linker allow of an We can the nicotinic acetylcholine receptor an for the structure of the GABAA receptor, as both belong to the superfamily of ligand-gated ion channels. The structure of the nicotinic acetylcholine receptor has been to A. Y. M. N. J. Mol. Biol. 1999; PubMed Scopus Google Scholar). All the of the superfamily a high sequence and the and it is assumed that also have a the of the receptor we can the minimal length of a the of one subunit to be the N is the This minimal length of is for the the receptor is but the N of the subunit of the tandem construct is the as the of the connection is to of either the N or the C from the of the linked subunits both in a of the required minimal length. the length of the subunit and the minimal length such a connection have to an subunit and the made to these we it that subunit is but we exclude this of the linker of the of acid of the first subunit to from the of the of the subunit to the β2 of in a In we combined the two tandem constructs and each with single γ subunits. In the of the tandem the channel the as type receptors, whereas in the of the tandem that of type receptors. The that both tandem constructs α-β and β-α in channels to was The for is thought to be the subunit E. Buhr A. Pharmacol. 1997; 18: Full Text PDF PubMed Scopus Google Scholar). This is in one of the two arrangements and in is that the subunit can benzodiazepine as it has been that receptors expressed from only β and γ subunits are to E. Baur R. G. Möhler H. Malherbe P. Neuron. 1990; 5: Full Text PDF PubMed Scopus Google Scholar). and likely is on a of one of the tandem constructs. We this for the In the of γ subunits, receptors containing α and β subunits alone are W. Lüddens H. Mol. Neurobiol. 1998; 18: 35-86Crossref PubMed Scopus (412) Google Scholar), but the γ subunit to a subunit to αβγ receptors Smart T.G. Moss S.J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, 18Tretter V. Ehya N. Fuchs K. Sieghart W. J. Neurosci. 1997; 17: 2728-2737Crossref PubMed Google Scholar). The of the tandem constructs with the γ subunits with the formation of or subunit the subunit of the tandem be In the of the tandem construct this in channels with to type receptors. In the tandem constructs have to to a β-α arrangement The linker now be and the of the subunits. is also that the Therefore, the is the for GABA and to be we the subunit arrangement βαγβα for the receptor. A as proposed for the tandem construct α-β in the of a γ2 subunit in subunit arrangements in the of a tetrameric receptor but subunit sequence in pentameric receptors composed of only α and β subunits. one of the tandem constructs in an which is be This arrangement can be from The of constructs containing the γ subunit and with the β-α tandem construct allow the study of the of single in one α or β it can be that subunits linked in a construct are able to also be to study the of subunit isoforms in the receptor In we have demonstrated the of subunit linkage for a ligand-gated ion the first time we have the minimal linker required for functional Our a pentameric structure of the GABAA receptor and exclude a This a for the study of subunit arrangement also of other ligand-gated ion channels. We V. for the