Hetero-oligomeric Assembly of P2X Receptor Subunits

P2X receptors are a distinct family of ligand-gated ion channels activated by extracellular ATP. Each of the seven identified subunit proteins (P2X1 through P2X7) has been reported to form functional homo-oligomeric channels when expressed in heterologous systems. Functional studies of native receptors, together with patterns of subunit gene expression, suggest that hetero-oligomeric assembly among members of this family may also occur. This prediction is supported by reports describing hetero-oligomeric assembly for three different recombinant subunit combinations. In this report, we systematically examined the ability of all members of the P2X receptor family to interact using a co-immunoprecipitation assay. The seven P2X receptor subunits were differentially epitope-tagged and expressed in various combinations in human embryonic kidney 293 cells. It was found that six of the seven subunits formed homo-oligomeric complexes, the exception being P2X6. When co-assembly between pairs of subunits was examined, all were able to form hetero-oligomeric assemblies with the exception of P2X7. Whereas P2X1, P2X2, P2X5, and P2X6 were able to assemble with most subunits, P2X3 and P2X4presented a more restricted pattern of co-association. These results suggest that hetero-oligomeric assembly might underlie functional discrepancies observed between P2X responses seen in the native and recombinant settings, while providing for an increased diversity of signaling by ATP. P2X receptors are a distinct family of ligand-gated ion channels activated by extracellular ATP. Each of the seven identified subunit proteins (P2X1 through P2X7) has been reported to form functional homo-oligomeric channels when expressed in heterologous systems. Functional studies of native receptors, together with patterns of subunit gene expression, suggest that hetero-oligomeric assembly among members of this family may also occur. This prediction is supported by reports describing hetero-oligomeric assembly for three different recombinant subunit combinations. In this report, we systematically examined the ability of all members of the P2X receptor family to interact using a co-immunoprecipitation assay. The seven P2X receptor subunits were differentially epitope-tagged and expressed in various combinations in human embryonic kidney 293 cells. It was found that six of the seven subunits formed homo-oligomeric complexes, the exception being P2X6. When co-assembly between pairs of subunits was examined, all were able to form hetero-oligomeric assemblies with the exception of P2X7. Whereas P2X1, P2X2, P2X5, and P2X6 were able to assemble with most subunits, P2X3 and P2X4presented a more restricted pattern of co-association. These results suggest that hetero-oligomeric assembly might underlie functional discrepancies observed between P2X responses seen in the native and recombinant settings, while providing for an increased diversity of signaling by ATP. Investigation of the native receptors mediating extracellular ATP signaling in tissues has been a difficult task due to the lack of useful pharmacological tools. For this reason, the cloning of ATP receptors (the P2 receptor family) and their recombinant expression has proven extremely useful in elucidating the basic properties of these proteins and for providing a template for further study into native P2 receptors. Two families of proteins mediating the actions of ATP have been identified: the metabotropic G protein-coupled P2Y receptors and the ionotropic P2X receptors (1Fredholm B.P. Abbracchio M.P. Burnstock G. Daly J.W. Harden T.K. Jacobson K.A. Leff P. Williams M. Pharmacol. Rev. 1994; 46: 143-156PubMed Google Scholar). The P2X receptors are nonselective ion channels thought to be oligomeric in nature, and they are expressed in many excitable and nonexcitable cells, where they mediate a variety of physiological actions, including smooth muscle contractility, neuroendocrine secretion, and modulation of synaptic transmission (2Burnstock G. Ciba Found. Symp. 1996; 198: 1-34PubMed Google Scholar,3Ralevic V. Burnstock G. Pharmacol. Rev. 1998; 50: 413-492PubMed Google Scholar). In addition, recent reports suggest that they may also play an important role in the transmission of pain perception (4Cook S.P. Vulchanova L. Hargreaves K.M. Elde R. McCleskey E.W. Nature. 1997; 387: 505-508Crossref PubMed Scopus (396) Google Scholar, 5Bardoni R. Goldstein P.A. Lee C.J. Gu J.G. MacDermott A.B. J. Neurosci. 1997; 17: 5297-5304Crossref PubMed Google Scholar). With the molecular identification of seven P2X receptor subunits, our understanding of the biophysical and pharmacological properties of these channels has been considerably increased. However, relatively little is known about the multimeric organization of this new class of channel receptors. Almost all known ionotropic receptors exist as hetero-oligomers (6Barnard E.A. Trends. Biochem. Sci. 1992; 17: 368-374Abstract Full Text PDF PubMed Scopus (147) Google Scholar). Importantly, their functional and pharmacological properties are directly determined by their subunit composition, with different subunit combinations yielding different phenotypes (e.g. see 7). This complexity appears to allow for diversity in signaling, and it therefore becomes material to determine whether each of the seven P2X receptor subunits is capable of co-assembling into hetero-oligomeric complexes as well. Studies using recombinant expression of individual subunits provide some evidence suggesting that in certain tissues, the native P2X response could be due to homo-oligomeric receptors (e.g. the P2X1 receptor and the P2X receptor found in the vas deferens) (8Valera S. Hussy N. Evans R.J. Adami N. North R.A. Suprenant A. Buell G. Nature. 1994; 371: 516-519Crossref PubMed Scopus (904) Google Scholar, 9Khakh B. Surprenant A. Humphrey P.P.A. Br. J. Pharmacol. 1995; 115: 177-185Crossref PubMed Scopus (57) Google Scholar). On the other hand, a P2X receptor phenotype seen in dorsal root sensory neurons appears to be the result of a hetero-oligomer containing P2X2 and P2X3subunits (4Cook S.P. Vulchanova L. Hargreaves K.M. Elde R. McCleskey E.W. Nature. 1997; 387: 505-508Crossref PubMed Scopus (396) Google Scholar, 10Lewis C. Neidhart S. Holy C. North R.A. Buell G. Surprenant A. Nature. 1995; 377: 432-435Crossref PubMed Scopus (896) Google Scholar). Additional evidence supporting the possible presence of hetero-oligomeric P2X receptors comes from Northern blot, in situ hybridization, and immunocytochemical studies that suggest that cell populations in a variety of tissues may express multiple subunit genes (11Vulchanova L. Riedl M.S. Shuster S.J. Buell G. Surprenant A. North R.A. Elde R. Neuropharmacology. 1997; 36: 1229-1242Crossref PubMed Scopus (382) Google Scholar, 12Collo G. North R.A. Kawashima E. Merlo-Pich E. Neidhart S. Surprenant A. Buell G. J. Neurosci. 1996; 16: 2495-2507Crossref PubMed Google Scholar, 13Nori S. Fumagalli L. Bo X. Bogdanov Y. Burnstock G. J. Vasc. Res. 1998; 35: 179-185Crossref PubMed Scopus (96) Google Scholar, 14Zhong Y. Dunn P.M. Xiang Z. Bo X. Burnstock G. Br. J. Pharmacol. 1998; 125: 771-781Crossref PubMed Scopus (55) Google Scholar, 15Xiang Z. Bo X. Burnstock G. Neurosci. Lett. 1998; 256: 105-108Crossref PubMed Scopus (200) Google Scholar, 16Collo G. Neidhart S. Kawashima E. Kosco-Vilbois M. North R.A. Buell G. Neuropharmacology. 1997; 36: 1277-1283Crossref PubMed Scopus (442) Google Scholar). If heteromultimerization is a common occurrence in the native setting, as each of the seven subunit proteins has a distinctive biophysical and pharmacological profile, then it becomes important to identify which subunits are capable of forming hetero-oligomeric assemblies and in which combinations. This is especially true if the hetero-oligomeric nature of such combinations is masked by the ability of a single subunit type to dominate the phenotype of the complex, a phenomenon seen in other ionotropic receptor families. If such a dominance was exhibited, it could be misleading, as it might affect only the obvious biophysical phenotype but not any underlying developmental and/or regulatory mechanism(s) and would thus prevent a more complete understanding of the physiological significance of ATP transmission via that hetero-oligomer. In order to address the question of subunit co-assembly for the P2X family, we tested for protein-protein interactions among members using a co-immunoprecipitation assay. We present here direct biochemical evidence for the preferential association between some but not all P2X subunits. This knowledge is essential not only in guiding studies to determine the molecular identities of endogenous P2X receptors, but also for understanding the molecular basis of interactions between the different subunits. The cDNAs for P2X1 and P2X3 were cloned from a rat heart library (kind gift of Dr. M. Tamkun, Vanderbilt University), and those for P2X4, P2X6, and P2X7 were cloned from a directional rat brain library in λZAP. The cDNAs encoding the P2X2 and P2X5 subunits were the kind gifts of Drs. D. Julius (University of California, San Francisco, CA) and G. Buell (Glaxo Research Institute, Geneva, Switzerland), respectively. Oligonucleotide primers (Life Technologies, Inc.) were designed to introduce the FLAG (DYKDDDDK) or the HA 1The abbreviations used are: HA, hemagglutinin; HEK 293, human embryonic kidney 293. (YPYDVPDYA) epitopes into the carboxyl termini of all P2X receptor subunits, immediately upstream of the stop codon. The P2X7 deletion mutant (P2X7(K419*) (asterisk indicates the stop codon)) was created by inserting the FLAG epitope immediately after amino acid 418 and replacing the lysine at amino acid position 419 with a stop codon by site-directed mutagenesis. All tagged P2X coding sequences were amplified by polymerase chain reaction using Vent polymerase (New England Biolabs) as described previously (17Torres G.E. Egan T.M. Voigt M.M. FEBS Lett. 1998; 425: 19-23Crossref PubMed Scopus (58) Google Scholar), digested with the appropriate restriction enzymes, and subcloned into the mammalian expression vector pRK-5 (18Schall T.J. Lewis M. Koller K. Lee A. Rice G.C. Wong G.H.W. Gatanaga T. Raab H. Kohr W.J. Goeddel D.V. Cell. 1990; 61: 361-370Abstract Full Text PDF PubMed Scopus (849) Google Scholar). All constructs were verified by DNA sequencing with the dideoxynucleotide chain termination method utilizing the deaza-T7 Sequenase kit from Amersham Pharmacia Biotech. HEK 293 cells were transiently transfected with epitope-tagged P2X receptor cDNAs individually or in different combinations. Cells were incubated with 1 μg of total cDNA and 6 μl of LipofectAMINE (Life Technologies, Inc.) in 1 ml of serum-free medium (Opti-MEM, Life Technologies, Inc.). After 5 h at 37 °C, the medium was replaced with complete minimal essential medium, and cells were incubated for 40–48 h. Whole cell current was recorded from single HEK 293 cells using AxoPatch 200 series amplifiers (Axon Instruments, Foster City, CA) and low resistance electrodes (1–2 MΩ). The typical holding voltage was −40 mV, at which the effect of ATP was generation of an inward current. Recording pipettes were filled with the following intracellular solution (in mm): 150 5 with The extracellular solution was as 150 1 with were by the and cell into the of of an of HEK 293 cells were into μl of proteins were to through from to by to Pharmacia The were in containing and incubated for 1 h with at or CA) After with a of containing were incubated with Pharmacia for 1 h. were in the and proteins were with the kit Pharmacia following the of HEK 293 cells in that been transfected with the cDNAs were three with and incubated at for 1 h in 1 1 containing the was using the or the in the presence of μl of G Pharmacia were with and in were for 5 and proteins were by by to These were then as described for the P2X receptors are nonselective channels by ATP. is that in order to form a ionotropic receptors be oligomeric be or hetero-oligomeric in a into the assembly of P2X subunits, we to determine whether each of the subunit proteins could form this we a co-immunoprecipitation in which the subunit was differentially epitope-tagged and the constructs were into HEK 293 cells. subunit was using the appropriate and the were then by to determine whether the subunit tagged with the other epitope was also result would that co-assembly we tagged all seven cloned subunits at their with the FLAG or HA in a total of different The of epitope for the of proteins that were of the or that these epitope-tagged subunits their and functional that the co-assembly results could be with by ATP in cells transfected with the various constructs were seen in of the tagged P2X subunits were functional and with properties to their In to this was a complete lack of from any of the P2X6 the We have to functional P2X6 receptors in transfected HEK 293 cells, and this is in with results reported by other as K. P. J. Neurosci. 1998; PubMed Google Scholar, M. C. Biochem. Res. 1996; PubMed Scopus Google Scholar). provide direct evidence that each P2X subunit homo-oligomeric the FLAG and HA tagged cDNAs for each of the subunits were as We then subunit and determined whether subunit was present in the by All proteins expressed and at when expressed in HEK 293 cells, with the exception of the FLAG and HA tagged constructs that were those of the other tagged P2X subunits. This of expression to the of as This that a to co-assembly be and for this reason, the are not described here but in a The of using the co-immunoprecipitation to study subunit co-assembly was by the that from cells individually transfected with each subunit not result in their co-immunoprecipitation in each subunit In addition, when any tagged subunits were from cells, their association was found to be to such as with or as is for P2X1 in A. These results from the and that tagged subunits could be and only by their and that was found between the the P2X2, P2X5, and P2X7 subunits form homo-oligomeric P2X6, the subunit that to in transfected cells, not form homo-oligomeric complexes This lack of co-immunoprecipitation is not due to low as the is present the and the is present in the of reaction These suggest that P2X6 subunits not assemble into and that this the of the P2X6 cDNA to provide in our transfected HEK 293 cells. the results the homo-oligomeric association of the various P2X receptor subunits, we to whether different P2X subunits could into hetero-oligomers when in HEK 293 cells. we different pairs of subunits and examined their ability to interact with each other using the co-immunoprecipitation the results of co-immunoprecipitation from cells with or the and The were with the and the complexes were by using the In proteins were suggesting that they into These interactions were to relatively including with or In addition, when the cells were transfected with the individual and together to the such interactions were not These results suggest that the were formed by and We then tested the ability of all subunits to in a and the results are in When the possible for were examined with the of P2X5, and P2X6 subunits but not the P2X7 receptor We then tested the ability of to interact with through with the of K.M. C. Surprenant A. North R.A. Kawashima E. J. Neurosci. 1997; 17: PubMed Google Scholar), with the of In addition, with and but to interact with of cell containing in the of but not or with with and The were also with and with and results were E. T. M. and M. M. All of these hetero-oligomeric interactions only in cells, and they were not when from cells individual subunits were and used for In addition, as for the homo-oligomeric these hetero-oligomeric were not by of or results that most P2X receptor subunits are capable of forming and hetero-oligomeric assemblies when in HEK 293 cells. The a in the of the co-immunoprecipitation we observed between and when from cells transfected with were to with the as as when This was a in to our results with the other six P2X subunits. We found that co-immunoprecipitation also when of were with those of the other subunits as with as in 5 it that when was the to be in the co-immunoprecipitation it formed interactions with the other subunits and we that these interactions were the result of such of present in the cells. a any of the subunits would with it of the For homo-oligomeric this would not be a as we that the subunit functional homo-oligomeric receptors However, for possible in hetero-oligomeric this would a for this of the study we used as the and the of the other subunits as This to the about by the of seen in 5 the with and the with we found from the for any from cells with together with any of the subunits were then seen in 5 interactions were observed for and or of were in from cells with However, was when was or suggesting that the observed interactions in the were in In to the subunits, and when with when the were it appears that in a restricted and only in forming assemblies with or from this study is the of P2X7 to form hetero-oligomeric the of the P2X7 receptor subunit is intracellular carboxyl we examined the that the lack of association between P2X7 and the other P2X subunits was due to an of that We a deletion mutant which was the amino acid and for ability to with different P2X subunits. in the P2X7 deletion mutant functional channels to those of the cDNA 6 was expressed at to the other subunits, and could be when expressed in HEK 293 cells of with or not result in their 6 This lack of association was not due to lack of expression for the subunits, as these proteins were in the reaction 6 the lack of association between P2X7 and the other P2X subunits not to the intracellular of P2X7. Almost all ion channels assemble as hetero-oligomeric complexes in which different subunits to channels with functional and this has been reported for three different combinations of P2X subunits C. Neidhart S. Holy C. North R.A. Buell G. Surprenant A. Nature. 1995; 377: 432-435Crossref PubMed Scopus (896) Google Scholar, K. P. J. Neurosci. 1998; PubMed Google Scholar, Egan Voigt M.M. Pharmacol. 1998; PubMed Scopus Google Scholar). has for the appropriate expression and of other ion channels for and/or In the of ligand-gated including those for and it has been that the properties of the recombinant hetero-oligomeric assemblies with those described for the endogenous channels E.A. P. H. G. C. Pharmacol. Rev. 1998; 50: Google Scholar, A. S. D. J. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, T. D. J. H. J. 1992; PubMed Scopus Google Scholar). This study is the to whether all known members of the P2X receptor subunit family form and hetero-oligomeric properties that have important the of ATP biochemical results that most P2X subunits are able to form homo-oligomeric assemblies with the from recombinant P2X receptors in HEK 293 cells or These protein-protein interactions were to receptor of or and are present only when subunits are into cells, suggesting that they are the result of or and not the result of interactions the was the lack of functional expression that we observed with the P2X6 an effect that appears to be by the of the P2X6 subunits to form homo-oligomeric assemblies as by the lack of of with are in to the describing this subunit in which HEK 293 cells were also used G. North R.A. Kawashima E. Merlo-Pich E. Neidhart S. Surprenant A. Buell G. J. Neurosci. 1996; 16: 2495-2507Crossref PubMed Google Scholar), but are in with from other that have examined expression using K. P. J. Neurosci. 1998; PubMed Google Scholar, M. C. Biochem. Res. 1996; PubMed Scopus Google Scholar). This lack of assembly and in our the that in cell of cells, of cells used at of or in other that are in the assembly of this subunit into but not a may provide an important for of functional P2X receptor in different cell and/or In situ studies suggest that different P2X receptor subunits are not only in the tissues but also may to the cells C. Neidhart S. Holy C. North R.A. Buell G. Surprenant A. Nature. 1995; 377: 432-435Crossref PubMed Scopus (896) Google Scholar, 12Collo G. North R.A. Kawashima E. Merlo-Pich E. Neidhart S. Surprenant A. Buell G. J. Neurosci. 1996; 16: 2495-2507Crossref PubMed Google Scholar, 13Nori S. Fumagalli L. Bo X. Bogdanov Y. Burnstock G. J. Vasc. Res. 1998; 35: 179-185Crossref PubMed Scopus (96) Google Scholar). These the that P2X receptor subunits might form hetero-oligomeric channel In the present we have a co-immunoprecipitation to whether the seven P2X receptor subunits with and if which combinations are in forming hetero-oligomeric results that hetero-oligomeric assembly and that it in a preferential We provide direct biochemical evidence for the and association of a of P2X subunit which are in In we not protein-protein interactions for or or for any containing P2X7 subunits. These of interactions the of using as a of the co-assembly of P2X subunits. The that P2X6 into hetero-oligomeric assemblies with many other subunits but not with the that P2X6 subunits might in a regulatory role hetero-oligomeric to has been for the and subunits of the channel L. Buell G. B. Nature. 1994; PubMed Scopus Google Scholar, C. P.M. J. 1998; Full Text Full Text PDF PubMed Scopus Google of observed P2X subunit co-assembly as determined using the co-immunoprecipitation described in a new Whereas most P2X receptor subunits with each other to form complexes, the P2X7 subunit to with any of the other P2X subunits. This lack of association not to the carboxyl of P2X7 a form of this receptor subunit not the or subunits. These results suggest that P2X7 subunits form homo-oligomeric hetero-oligomeric Two recent studies of native our a by L. S.J. J. 1998; Full Text Full Text PDF PubMed Scopus (57) Google identified populations of ATP receptors in cells, the properties of which with those for homo-oligomeric and and C. G. A. P. Res. 1998; PubMed Scopus Google provide suggesting that P2X1 and P2X7 receptors are expressed as homo-oligomeric populations in smooth muscle cells. from the biochemical to functional studies is in order to determine the physiological significance of P2X subunit are reports describing native receptors that not with the individual subunits expressed suggesting that hetero-oligomeric assembly might result in the generation of a The such study P2X co-assembly was for P2X2 and which were to form a hetero-oligomeric channel when in HEK 293 cells C. Neidhart S. Holy C. North R.A. Buell G. Surprenant A. Nature. 1995; 377: 432-435Crossref PubMed Scopus (896) Google Scholar). This phenotype with the receptors described in some sensory neurons where the proteins are suggesting that this in the native as well. Additional evidence for hetero-oligomeric assembly has the of this K. P. J. Neurosci. 1998; PubMed Google functional evidence for a channel formed between epitope-tagged and P2X6 subunits, and we reported that the of P2X1 and P2X5 subunits also in a channel with a phenotype Egan Voigt M.M. Pharmacol. 1998; PubMed Scopus Google Scholar). For pairs of subunits, is a pattern of suggesting that such combinations might be of native receptors G. North R.A. Kawashima E. Merlo-Pich E. Neidhart S. Surprenant A. Buell G. J. Neurosci. 1996; 16: 2495-2507Crossref PubMed Google Scholar). the in this study provide a for into the of recombinant and native hetero-oligomeric P2X receptor It be a that of subunits result in the generation of a receptor This is when results from this study are in with those by Lewis C. Neidhart S. Holy C. North R.A. Buell G. Surprenant A. Nature. 1995; 377: 432-435Crossref PubMed Scopus (896) Google Scholar). In that study of P2X subunit it was reported that of and not to the of receptors with a that was to that the subunits not our results also provide evidence that and not we found that the subunit This when together with the biophysical C. Neidhart S. Holy C. North R.A. Buell G. Surprenant A. Nature. 1995; 377: 432-435Crossref PubMed Scopus (896) Google Scholar), that lack of a phenotype not that subunits are not to form a hetero-oligomeric channel is that when receptor subunits to form a complex, subunit type dominate the phenotype of the and thus only a single phenotype is This phenomenon is observed in the ionotropic receptor family, where the presence of the subunit hetero-oligomeric receptor assemblies with to and properties Neurosci. 16: Full Text PDF PubMed Scopus Google Scholar). this if it is the of subunits in an assembly that the observed and a of hetero-oligomeric receptor channels containing different of the subunits form after a then it is possible that populations of the homo-oligomeric receptors, would be This is was observed by C. Neidhart S. Holy C. North R.A. Buell G. Surprenant A. Nature. 1995; 377: 432-435Crossref PubMed Scopus (896) Google for the if is effect the biophysical phenotype of a hetero-oligomeric assembly could have important receptor by the of a to intracellular or a an to hetero-oligomeric assembly the functional properties of these we are the functional of the interactions from this biochemical provide to elucidating the subunit of endogenous P2X receptors. In addition, the of the interactions among the members of the P2X receptor family provide a useful in the molecular in hetero-oligomeric P2X receptor results that could have these