Forced Subunit Assembly in α1β2γ2 GABAAReceptors

Abstract

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 a containing the of the the γ2 the and the of the This vector was with in and for at The (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 a containing the of the of the β the and the α The two were and was by The construct was made from (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 The triple constructs from and from and and from and were prepared The have been were from the containing the different tandem or triple constructs or the single α1, β2, and and from the vector J. 1999; PubMed Scopus Google Scholar) a channel A of about residues was to each The of the was on a for of the and of as on the same combinations of or and were in and at For the was and the were in of oocytes from the of the of and were as described E. J. PubMed Scopus Google Scholar). were with of the For combinations of the triple constructs with the tandem of and were The combination of single α1, β2, and γ2 subunits was expressed at allow of expressed for the channel was to a of The oocytes were in modified at 18 for the All were in containing and at a of For the of was for were by a from a of to the of of the receptor channel and the channel the of the two channels not with each The was to the of the same The of at oocytes subunit combination was with the wild type of the were by of by diazepam was at a of the in combination with of diazepam and expressed as 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 α and β subunits of the GABAA receptor 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). linked constructs to a possible arrangement of subunits in the α1β2γ2 However, some uncertainty to a possible of dual subunit constructs. We have linked three subunits in different and expressed in combination with tandem constructs. Triple subunit constructs dual subunit constructs for In the following, the of subunits in subunit constructs is described as the of the subunit linked to the N-terminal of the the γ subunit to of the tandem constructs and linker lengths for the to be test of these dual constructs γ-α, β-γ, or were with α-β or β-α constructs and single α or β subunits to receptors of the not The required linker lengths for functional were found to be amino acid residues for γ-β, amino acid residues for and residues for For a tandem to amino acid residues in length were were found to result only in functional channel expression. In the following, the triple constructs and were the of and in a is different arrangements of and γ subunits are possible this stoichiometry we arrangements containing one γ and two each of α and β in an of al. V. Ehya N. Fuchs K. Sieghart W. J. Neurosci. 1997; 17: 2728-2737Crossref PubMed Google Scholar) an arrangement of α and γ or β and γ to dimers In these a β or α subunit is to of α and in contrast, to the formation of and arrangements are and They are the most arrangements as to the inferred two αβ and αγ subunit required for the formation of two and one benzodiazepine-binding the subunits are not only one of these two arrangements is to form the subunit for triple construct we show that the arrangement is not functional This was not the which that of the α-β construct with single γ subunits in as with wild type receptors. did not result in functional channels upon in combination with or γ-α, or in combination with α or β In contrast, could be with β-α be by or α-β by β-α by for several combinations of triple constructs with tandem constructs shown to result in the functional were to the of the channel expressed in the same were in each in oocytes from two different of all to a channel with the subunit arrangement the functional of channels from different combinations of triple and dual subunit constructs. All these combinations and result in the identical arrangement of subunits around the and can be as of the of the around the five subunits was expressed in combination with β-α at a stoichiometry of only to about of of the upon of subunits and γ at were for and β-α expressed at The for this for is not receptors with a different stoichiometry arrangement from the one were also for functional expression. Triple constructs and 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 did not result in functional channel expression. From the of by of we can also exclude the arrangements and results make it that is the functional arrangement of the The that no or were linker length and for of the subunit combinations that proteolysis in the linker is not to a as for for and can be thought to channels. channels are not are not formed to a receptors with the subunit arrangement made from linked constructs we investigated properties to the agonist and the competitive antagonist we the properties of the tandem construct β-α in combination with single γ2 subunits. with receptors made from single subunits we a A similar been 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). three of four combinations of triple constructs with tandem constructs that were functional were characterized in A with the subunit All of properties similar to wild type receptors and was only and an of in these competitive antagonist, and benzodiazepine properties of arranged receptors made from different combinations of triple and dual subunit constructs. The were from oocytes from different of oocytes are given as in a a of by the competitive of on all subunit construct combinations resulting in functional receptors The were similar to those of the receptor made from single subunits. the agonist and competitive antagonist properties of the different constructs. From these results we can that the used here between the subunits have on the for channel and the by has been that by diazepam of by in oocytes is and in oocytes A.J. Baur R. Sigel E. Czajkowski C. PubMed Scopus Google Scholar). Therefore, it was to receptors formed from triple and tandem constructs in this The to of diazepam did not from receptors containing subunits the of However, was a and in different oocytes of this by diazepam of by in oocytes at about and was in different a of about with was the γ2 subunit was linked to other subunits to this by diazepam in oocytes or linked constructs The combinations and also for which were those found for wild type receptors and not has been that the by diazepam in oocytes with for and γ A.J. Baur R. Sigel E. Czajkowski C. PubMed Scopus Google Scholar). This not to in oocytes linked subunits in and of the by diazepam in and linked receptors. The a were in for by diazepam The was were with a are in receptors and in linked In the present study we used linked subunits of the GABAA receptor to study the arrangement of subunits in α1β2γ2 receptors. We receptors made from combinations of triple and dual subunit constructs containing α1, β2, in different We present evidence that α1β2γ2 receptors have a subunit and arrangement of from N-terminal to should be that we to at the of the subunit combinations to form functional ion channels. For combinations that did not result in we at present whether this is to of constructs, or to assembly or whether the receptors the and are to The of GABAA receptors and γ subunits D. Fritschy J.M. Trzeciak A. Bannwarth W. Möhler H. J. Biol. Chem. 1994; 269: 27100-27107Abstract Full Text PDF PubMed Google Scholar, J.M. D. S. T. H. Proc. Natl. Acad. Sci. U. S. A. 1992; PubMed Scopus Google Scholar, E. 1994; Google Scholar, J.M. H. J. 1995; PubMed Scopus Google Scholar, P. Fritschy J.M. D. Sieghart W. 1996; PubMed Scopus Google Scholar). This subunit can be by three subunits of one type with one subunit each of the other two or two subunits each from two with one subunit of the The former stoichiometry has been for and α1β2γ2 receptors by 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 stoichiometry receptors with and have been A stoichiometry is by Y. Wang R. Barot S. Weiss D.S. J. Neurosci. 1996; 16: 5415-5424Crossref PubMed Google Scholar), V. Ehya N. Fuchs K. Sieghart W. J. Neurosci. 1997; 17: 2728-2737Crossref PubMed Google Scholar), and 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 that also two γ subunits can the same pentamer. This has been suggested on the of of isoforms of the γ subunit the same receptor and have been in A. J. 1996; PubMed Scopus Google Scholar, K. Whiting P.J. McKernan R.M. Mol. Pharmacol. 1994; Google Scholar). We found that only combinations of triple and dual constructs, which in the arrangement containing and subunit(s), were functionally expressed with properties similar to receptors made from single subunits. of subunits linked subunit constructs that result in a different stoichiometry or a different arrangement were not study is to α1, β2, and receptors. is not clear whether receptors containing different isoforms of these subunit have the same stoichiometry and arrangement as the that for the formation of GABA-binding sites defined be it is that all and 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, W. Seeburg P.H. 1992; PubMed Scopus (231) Google Scholar) receptors the same we expressed triple constructs in combination with tandem constructs to form receptors we properties of the resulting receptors similar to receptors made from subunits. The between the subunits not in one did we a in the This is in the of as with for all other receptors This observation a of the receptor al. J. Czajkowski C. Neurosci. 2001; PubMed Scopus Google Scholar) suggested an of the subunits upon channel They investigated αβ receptors and that subunits and in the same subunit in the This β subunit can be thought to be by the γ subunit in the In this the linker between γ and β in the construct could the suggested of the linked subunits, which could to the of the This however, did not to be in the dual construct when expressed in combination with the triple construct A dual subunit construct be a triple construct. of a linker the triple construct could the For all receptors formed from linked subunits we a to the in the with receptors made from single subunits. one this could be to or properties of the receptor by the other it has been that of single and γ subunits in oocytes or to a of and αβ receptors A.J. Baur R. Sigel E. Czajkowski C. PubMed Scopus Google Scholar). The of αβ receptors is about that of receptors about E. Baur R. J. PubMed Scopus Google Scholar). of linked constructs not allow the formation of pentameric αβ and the of the to the could at be to of receptors. This is supported by the of by diazepam of receptors made from linked subunits. of αβ in receptors diazepam A.J. Baur R. Sigel E. Czajkowski C. PubMed Scopus Google Scholar). to the of subunits in the receptor and not allow a about the absolute the when viewed from the synaptic cleft. The 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) to an the absolute of the extracellular of the subunits and of the nicotinic acetylcholine The acetylcholine-binding protein is a protein homologous to the extracellular domain of the nicotinic acetylcholine receptor of The GABAA receptor to the same superfamily of ligand-gated ion channels and is homologous to the nicotinic acetylcholine The of amino acid residues that form agonist and benzodiazepine-binding respectively 27Sigel E. Buhr A. Trends Pharmacol. Sci. 1997; 18: 425-429Abstract Full Text PDF PubMed Scopus (345) Google Scholar) to on which of the α subunit γ and β subunits are when viewed from the synaptic cleft. Therefore, the absolute arrangement of the α1β2γ2 receptor is from the to the in when viewed from the synaptic This work will allow the study of the of site located on an α or β which was far were two sites on the two subunits by a mutation in GABAA receptors. The of a forced subunit assembly will of a mutation in only one This for allow of the two agonist sites located at the αβ subunit Forced subunit assembly will have on the of receptor containing different isoforms of the same subunit that are made of or different subunits be by of the of the single subunits different receptor can be of of subunits in triple and tandem constructs will allow the study of properties of defined receptor the GABAA receptor to a superfamily of ligand-gated ion channels, methods described here are applicable to and nicotinic acetylcholine and 5HT3 receptors. We van for in the linked subunit constructs and V. for the