Hetero-oligomeric Assembly of P2X Receptor Subunits
SPECIFICITIES EXIST WITH REGARD TO POSSIBLE PARTNERS*

Gonzalo E. Torres, Terrance M. Egan, and Mark M. VoigtDagger

From the Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, St. Louis, Missouri 63104

    ABSTRACT
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Abstract
Introduction
References

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 P2X4 presented 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.

    INTRODUCTION
Top
Abstract
Introduction
References

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 (1). 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 (2, 3). In addition, recent reports suggest that they may also play an important role in the transmission of pain perception (4, 5). 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 (6). 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) (8, 9). 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 P2X3 subunits (4, 10). 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 (11-16). 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.

    EXPERIMENTAL PROCEDURES

DNA Constructs-- 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 lambda ZAP. 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 HA1 (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 (17), digested with the appropriate restriction enzymes, and subcloned into the mammalian expression vector pRK-5 (18). All constructs were verified by DNA sequencing with the dideoxynucleotide chain termination method utilizing the deaza-T7 Sequenase kit from Amersham Pharmacia Biotech.

Transfection of HEK 293 Cells-- 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.

Electrophysiological Recordings-- 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 MOmega ). 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 CsCl, 10 tetraethylammonium-Cl, 5 EGTA, 10 HEPES, pH 7.3, with CsOH. The extracellular solution was as follows: 150 mM NaCl, 1.0 mM CaCl2, 1 mM MgCl2, 10 mM glucose, 10 mM HEPES, pH 7.3, with NaOH. Drugs were applied by manually moving the electrode and attached cell into the line of flow of solutions exiting an array of inlet tubes.

Western Blot Analysis-- HEK 293 cells grown on 35-mm culture plates were scraped into 100 µl of sodium dodecyl sulfate sample buffer. Solubilized proteins were subjected to SDS-polyacrylamide gel electrophoresis through gels ranging from 8 to 14% polyacrylamide, followed by transfer to Hybond-ECL membranes (Amersham Pharmacia Biotech). The filters were blocked overnight in 20 mM Tris, pH 7.6, 145 mM NaCl, 0.05% Tween 20 containing 5% nonfat dry milk and incubated for 1 h with primary antibody (M2 anti-FLAG (Kodak) at 10 µg/ml, or anti-HA (BABCo, Berkeley, CA) 1:1000). After several washes with a buffer of 20 mM Tris, pH 7.6, 145 mM NaCl, 0.05% Tween 20 containing 5% nonfat dry milk, filters were incubated with peroxidase-conjugated sheep anti-mouse antibody (Amersham Pharmacia Biotech) for 1 h. Filters were washed extensively (24-72 h) in the incubation buffer, and immunoreactive proteins were detected with the ECL detection kit (Amersham Pharmacia Biotech) following the manufacturer's suggestions.

Immunoprecipitations-- Monolayers of HEK 293 cells in 35-mm dishes that had been transfected with the indicated cDNAs were washed three times with phosphate-buffered saline and incubated at 4 °C for 1 h in solubilization buffer (phosphate-buffered saline, 1 mM phenylmethylsulfonyl fluoride, 1 mM (4-(2-aminoethyl)benzenesulfonyl fluoride-HCl, 10 µg/ml leupeptin) containing the indicated detergent. Immunoprecipitation was carried out using either the M2 anti-FLAG antibody (5 µg/ml) or the anti-HA antibody (1:500) in the presence of 50 µl of protein G Gamma-Bind agarose (Amersham Pharmacia Biotech). Immunoprecipitates were washed five times with solubilization buffer and resuspended in protein sample buffer. Samples were boiled for 5 min, and proteins were analyzed by SDS-PAGE, followed by transfer to nitrocellulose filters. These blots were then treated as described for the Western blots.

    RESULTS

P2X receptors are nonselective cation channels gated by ATP. Current thinking is that in order to form a transmembrane ion-conducting pore, ionotropic receptors must be oligomeric proteins. Such oligomers can be either homo- or hetero-oligomeric in nature. As a first step into investigating the assembly of P2X subunits, we wanted to determine whether each of the subunit proteins could indeed form homo-oligomers. To test this hypothesis, we utilized a co-immunoprecipitation assay in which the same subunit was differentially epitope-tagged and the resulting two constructs were co-transfected into HEK 293 cells. Subsequently, one subunit was immunoprecipitated using the appropriate anti-tag antibody, and the immunoprecipitates were then analyzed by Western blot to determine whether the subunit tagged with the other epitope was also present. A positive result would indicate that co-assembly had occurred. Therefore, we tagged all seven cloned subunits at their C terminus with either the FLAG or HA epitopes, resulting in a total of 14 different constructs. The use of epitope tags allowed for the unequivocal detection of specific proteins that were of the same or similar size.

To ensure that these epitope-tagged subunits retained their structural and functional integrity so that the co-assembly results could be interpreted with confidence, whole-cell currents elicited by ATP in cells transfected with the various constructs were measured. As seen in Fig. 1, 12 of the 14 tagged P2X subunits were functional and gave ATP-induced currents with kinetic properties similar to their wild-type parents. In contrast to this was a complete lack of activity from any of the P2X6 constructs, even the wild-type. We have consistently failed to observe functional P2X6 receptors in transfected HEK 293 cells, and this is in keeping with results reported by two other groups as well (19, 20).


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Fig. 1.   ATP-gated currents through tagged P2X receptors resemble their wild-type counterparts. Each panel shows three superimposed currents evoked from cells transfected with either the wild-type, FLAG-tagged, or HA-tagged constructs of individual P2X subunits. Peak currents are normalized to show that the time courses of the ATP-gated currents are not affected by addition of either tag. ATP was applied at a concentration of 30 µM for P2X1-5 and 300 µM for P2X7. Holding voltage was -40 mV. No current was observed from cells transfected with either wild-type or tagged P2X6 cDNAs. P2X1·HA and P2X7·FLAG were the only constructs that gave currents consistently smaller than their wild-type parents, and this resulted in the higher levels of noise seen for those traces.

Homo-oligomeric Assembly of P2X Subunits-- To provide direct evidence that each P2X subunit can undergo homo-oligomeric assembly, the FLAG and HA tagged cDNAs for each of the P2X1-3 and P2X5-7 subunits were co-transfected as pairs. We then immunoprecipitated one subunit (for instance, P2X1·FLAG) and determined whether its cognate subunit (P2X1·HA) was present in the immunoprecipitate by Western blot. All proteins expressed robustly and at qualitatively equivalent levels when expressed in HEK 293 cells, with the exception of P2X4. Both the FLAG and HA tagged P2X4 constructs gave protein levels that were remarkably higher (more than 10 times higher) than those of the other tagged P2X subunits. This high level of expression lead to the appearance of nonspecific interactions, as demonstrated by in vitro mixing experiments (see below). This factor required that a slightly altered approach to investigating P2X4 co-assembly be used, and for this reason, the P2X4 data are not described here but in a section presented under "Results."

The validity of using the co-immunoprecipitation approach to study subunit co-assembly was demonstrated by the fact that simply mixing lysates from cells individually transfected with each subunit did not result in their co-immunoprecipitation (Fig. 2, left lane in each subunit panel). In addition, when any two tagged subunits were co-immunoprecipitated from co-transfected cells, their association was found to be resistant to strong solubilizing conditions such as 1% Triton with 500 mM NaCl, or 5% Triton, as is shown for P2X1 in Fig. 2A. These results from the mixing and co-transfection experiments demonstrated that tagged subunits could be efficiently and selectively immunoprecipitated only by their respective anti-tag antibody and that no detectable cross-reactivity was found between the two antibodies.


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Fig. 2.   All P2X subunits except P2X6 form homo-oligomeric complexes. A, HEK 293 cells were transfected with P2X1·FLAG and P2X1·HA cDNAs and lysed with various detergents (lanes 1 and 2, 1% Nonidet P-40; lane 3, 1% Triton plus 0.5 M NaCl; and lane 4, 5% Triton). Solubilized proteins were immunoprecipitated with the anti-FLAG antibody and detected by Western blot with the anti-HA antibody. For mixing experiments, lysates from cells expressing individual subunits were mixed, immunoprecipitated with anti-FLAG antibody and detected with anti-HA antibody (lane 1). B, cells expressing P2X2·FLAG/P2X2·HA, P2X3·FLAG/P2X3·HA, P2X5·FLAG/P2X5·HA, and P2X7·FLAG/P2X7·HA combinations, as indicated, were lysed with 1% Nonidet P-40 and analyzed as described above. In each blot, the left lane represents the mixing experiment between the FLAG-tagged subunit and the HA-tagged subunit, whereas the right lane corresponds to the co-expression of both subunits. C, mixing (left lane) or co-expression (right lane) of P2X6·FLAG and P2X6·HA subunits did not result in co-immunoprecipitation. For the co-expression experiment, the blot was reprobed with the anti-FLAG antibody and the supernatant was immunoprecipitated and probed with the anti-HA antibody to demonstrate the presence of P2X6·FLAG and P2X6·HA, respectively.

As Fig. 2B illustrates, the P2X2, P2X3, P2X5, and P2X7 subunits did form homo-oligomeric assemblies. P2X6, the subunit that failed to give ATP-induced currents in transfected cells, did not form homo-oligomeric complexes (Fig. 2C, left panel). This lack of co-immunoprecipitation is not due to low protein levels, as the P2X6·FLAG protein is present on the blot and the P2X6·HA protein is present in the supernatant of precipitation reaction (Fig. 2C, right panel). These findings suggest that P2X6 subunits do not assemble into homo-oligomers and that this failure underlies the inability of the P2X6 cDNA to provide ATP-gated currents in our transfected HEK 293 cells.

Hetero-oligomeric Assembly of P2X Subunits-- Because the results obtained above demonstrated the homo-oligomeric association of the various P2X receptor subunits, we wanted to next examine whether different P2X subunits could co-assemble into hetero-oligomers when co-expressed in HEK 293 cells. To test this, we co-transfected different pairs of P2X·FLAG/P2X·HA subunits and examined their ability to interact with each other using the co-immunoprecipitation technique. Fig. 3 shows the results of co-immunoprecipitation experiments carried out from cells co-expressing P2X1·FLAG with P2X2·HA, or the converse pair, P2X1·HA and P2X2·FLAG. The detergent-solubilized membranes were immunoprecipitated with the anti-FLAG antibody, and the resulting complexes were analyzed by Western blot using the anti-HA antibody. In both instances, both proteins were immunoprecipitated, suggesting that they had co-assembled into hetero-oligomers. These interactions were again resistant to relatively harsh solubilization conditions, including 1% Triton with 500 mM NaCl or 5% Triton. In addition, when the cells were transfected with the individual cDNAs, solubilized, and mixed together prior to the immunoprecipitation, such interactions were not detected. These results suggest that the co-assemblies were formed by specific and stable subunit-subunit interactions.


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Fig. 3.   Hetero-oligomeric interaction between epitope-tagged P2X1 and P2X2 subunits. HEK 293 cells were transfected with P2X1·FLAG/P2X2·HA (lanes 1-4) or with P2X2·FLAG/P2X1·HA (lanes 5-8) combinations and lysed with a buffer containing 1% Nonidet P-40 (lanes 2 and 6), 1% Triton plus 0.5 M NaCl (lanes 3 and 7) or 5% Triton (lanes 4 and 8). FLAG-tagged subunits were immunoprecipitated with the anti-FLAG antibody, and HA-tagged subunits were detected by Western blot with the anti-HA antibody. Mixing experiments were performed from cells expressing individual subunits (P2X1·FLAG/P2X2·HA (lane 1) or P2X2·FLAG/P2X1·HA (lane 5)) and solubilized with 1% Nonidet P-40 prior to immunoprecipitation.

We then tested the ability of all subunits to co-assemble in a pairwise fashion and the results are shown in Fig. 4. When the possible partners for P2X1·FLAG were examined (Fig. 4A), immunoprecipitation with the anti-FLAG antibody caused co-precipitation of HA-tagged P2X3, P2X5, and P2X6 subunits but not the HA-tagged P2X7 receptor subunit. We then tested the ability of P2X2·FLAG to interact with P2X3·HA through P2X7·HA (Fig. 4B). Consistent with the findings of Radford et al. (21), immunoprecipitation with the anti-FLAG (P2X2) antibody caused co-precipitation of P2X3-HA. In addition, P2X2·FLAG co-precipitated with P2X5·HA, and P2X6·HA but failed to interact with P2X7·HA. Immunoprecipitation of co-transfected cell lysates containing P2X3·FLAG resulted in the detection of P2X5·HA but not P2X6·HA or P2X7·HA (Fig. 4C). Similarly, P2X5·FLAG co-precipitated with P2X6·HA, whereas neither P2X5·FLAG nor P2X6·FLAG associated with P2X7·HA (Fig. 4, D and E). The reverse experiments were also performed (i.e., immunoprecipitation with anti-HA and probing with anti-FLAG), and identical results were obtained.2 All of these hetero-oligomeric interactions occurred only in co-transfected cells, and they were not detectable when lysates from cells expressing individual subunits were mixed and used for immunoprecipitation. In addition, as for the homo-oligomeric assemblies, these hetero-oligomeric co-assemblies were not disrupted by high concentration of salt or detergent.2These results demonstrate that most P2X receptor subunits are capable of forming specific and stable hetero-oligomeric assemblies when co-expressed in HEK 293 cells.


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Fig. 4.   Hetero-oligomeric interaction among P2X receptor subunits. HEK 293 cells were transfected with pairs of FLAG- and HA-tagged subunits as indicated and lysed with 1% Nonidet P-40. P2X1·FLAG (A), P2X2·FLAG (B), P2X3·FLAG (C), P2X5·FLAG (D), and P2X6·FLAG (E) were immunoprecipitated with the anti-FLAG antibody, and the respective HA-tagged subunits were detected by Western blot with the anti-HA antibody. For each blot, the left lane represents the mixing experiment, whereas the right lane corresponds to the co-transfection of subunits. The heavy bar on the right side of each blot represents the expected size of the HA-tagged subunit. For the combinations that did not result in co-immunoprecipitation, analysis of the blots and supernatants demonstrated that the two tagged subunits had been translated (data not shown).

Special Considerations Required for P2X4 Co-assembly-- The P2X4 protein presented a difficulty in the interpretation of the co-immunoprecipitation experiments. To begin with, we observed interaction between P2X4·FLAG and P2X4·HA even when lysates from cells transfected with either construct alone were combined prior to immunoprecipitation with the anti-FLAG antibody, as well as when co-transfected (Fig. 5A, left panel). This was a finding in marked contrast to our results with the other six P2X subunits. We found that nonspecific co-immunoprecipitation also occurred when lysates of P2X4·HA were mixed with those of the other FLAG-tagged subunits as well (e.g., with P2X1·FLAG, as shown in Fig. 5A, right panel). Thus, it appeared that when P2X4·HA was the partner to be detected in the co-immunoprecipitation experiments, it formed nonspecific interactions with the other subunits present, and we surmised that these interactions were the result of having such high levels of P2X4 protein present in the cells. As a result, precipitating any of the FLAG-tagged subunits would invariably bring with it appreciable amounts of the P2X4·HA. For homo-oligomeric assembly, this would not be a problem as we already know that the P2X4 subunit forms functional homo-oligomeric receptors (Fig. 1). However, for investigating possible partners in hetero-oligomeric assembly, this would pose a problem. Thus, for this portion of the study we used P2X4·FLAG as the immunoprecipitation target and the HA-tagged versions of the other subunits as test partners. This approach seemed to circumvent the artifacts brought about by the high levels of P2X4 protein. As seen in Fig. 5B, performing the immunoprecipitations with P2X4·FLAG and probing the blots with anti-HA, we found no detectable co-immunoprecipitations from the mixed lysates for any subunit. Immunoprecipitations from cells co-transfected with P2X4·FLAG together with any one of the HA-tagged subunits were then carried out. As seen in Fig. 5B, no detectable interactions were observed for P2X4·FLAG and P2X1·HA, P2X3·HA, or P2X7·HA. Small amounts of P2X2·HA were detected in precipitates from co-transfected cells solubilized with 1% Nonidet P-40. However, no P2X2·HA was detected when solubilization was carried out under stronger conditions (1% Triton plus 500 mM NaCl, or 5% Triton), suggesting that the observed interactions in the 1% Nonidet P-40 were nonspecific in nature. In contrast to the above-mentioned subunits, P2X4·HA, P2X5·HA, and P2X6·HA gave strong signals when co-expressed with P2X4·FLAG, even when the harsher solubilization conditions were used. Thus, it appears that P2X4·FLAG co-assembles in a restricted fashion and only participates in forming stable assemblies with itself, P2X5·HA or P2X6·HA.


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Fig. 5.   P2X4 subunits form homo-oligomeric receptors and participate in heteromeric assemblies containing P2X5 or P2X6 subunits. A, HEK 293 cells were transfected either individually or together with P2X4·FLAG/P2X4·HA (left panel) or P2X1·FLAG/P2X4·HA (right panel) and solubilized with 1% Nonidet P-40. FLAG-tagged subunits were immunoprecipitated with anti-FLAG antibody and P2X4·HA detected by Western blot with anti-HA antibody. Note that the interaction between P2X1·FLAG and P2X4·HA was observed regardless of whether the two subunits were mixed (right panel, left lane) or co-expressed (right lane) prior to immunoprecipitation. B, HEK 293 cells transfected with the following combinations P2X4·FLAG/P2X1·HA, P2X4·FLAG/P2X2·HA, P2X4·FLAG/P2X3·HA, P2X4·FLAG/P2X5·HA, P2X4·FLAG/P2X6·HA, and P2X4·FLAG/P2X7·HA were lysed with 1% Nonidet P-40, and solubilized proteins were immunoprecipitated with anti-FLAG antibody and detected by Western blot with anti-HA antibody. For the P2X4·FLAG/P2X2·HA combination, the left lane is from mixed lysates, and the other three lanes (from left to right) are membranes from co-transfected cells treated with (i) 1%Nonidet P-40, (ii) 1% Triton X-100/0.5 M NaCl, and (iii) 5% Triton X-100. For all other combinations, the left lane is from mixed lysates, and the right lane is from co-transfected cells. The heavy bar on the right side of each blot represents the expected size of the HA-tagged subunit.

Role of the C Terminus in P2X7 Co-assembly-- Another striking finding from this study is the inability of P2X7 to form hetero-oligomeric assemblies. Because the main structural difference of the P2X7 receptor subunit is its long intracellular carboxyl terminus, we examined the possibility that the lack of association between P2X7 and the other P2X subunits was somehow due to an intrinsic property of that domain. We generated a deletion mutant (P2X7(K419*)), which was lacking the last 177 amino acid residues, and assayed for its ability to co-associate with several different P2X subunits. As shown in Fig. 6, the FLAG-tagged P2X7 deletion mutant generated functional channels similar to those of the wild-type cDNA (Fig. 6A), was expressed at levels comparable to the other subunits, and could be efficiently immunoprecipitated when expressed in HEK 293 cells (Fig. 6B). Co-expression of P2X7(K419*)FLAG with P2X1·HA, P2X2·HA, or P2X3·HA did not result in their co-precipitation (Fig. 6C). This lack of association was not due to lack of expression for the HA-tagged subunits, as these proteins were detectable in the reaction supernatant (Fig. 6D). Thus, the lack of association between P2X7 and the other P2X subunits does not appear to involve the long intracellular tail of P2X7.


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Fig. 6.   P2X7 lacking its long intracellular C terminus does not form heteromeric assemblies. A, 1 mM ATP evoked an inward current in cells transfected with P2X7·FLAG(K419*). Holding voltage was -40 mV. B, HEK 293 cells were transfected without (control (C), left lane) or with (right lane) P2X7·FLAG(K419*) cDNA and lysed with 1% Nonidet P-40, and solubilized proteins were immunoprecipitated and detected by Western blot with anti-FLAG antibody. C, mixing and co-expression experiments from cells transfected with P2X7·FLAG(K419*) (shown as Delta 7) and the following subunits: P2X1·HA (left panel), P2X2·HA (middle panel), and P2X3·HA (right panel). D, analysis of the supernatants from cells co-expressing the constructs in C, demonstrates the presence of P2X1·HA, P2X2·HA, and P2X3·HA. The heavy bar on the right side of each blot represents the expected size of the HA-tagged subunit.


    DISCUSSION

Almost all ion channels assemble as hetero-oligomeric complexes in which different subunits associate to generate channels with unique functional properties, and this has been reported for three different pair-wise combinations of P2X subunits (10, 19, 22). Subunit composition has proved critical for the appropriate expression and function of other ion channels for particular tissue and/or cellular contexts. In the case of ligand-gated channels, including those for acetylcholine, glutamate, GABA, and glycine, it has been shown that the properties of the recombinant hetero-oligomeric assemblies correlate well with those described for the endogenous channels (7, 23-25). This study is the first to investigate whether all known members of the P2X receptor subunit family can form homo- and hetero-oligomeric assemblies, properties that can have important influences on the characteristics of ATP signaling.

Our biochemical results demonstrate that most P2X subunits are able to form homo-oligomeric assemblies consistent with the electrophysiological data obtained from recombinant P2X receptors in either HEK 293 cells or Xenopus oocytes. These protein-protein interactions were sufficiently strong to withstand receptor solubilization under conditions of high salt or detergent concentrations, and are present only when subunits are co-transfected into cells, suggesting that they are the result of specific co- or posttranslational processing mechanisms and not the result of random interactions within the plasma membrane per se. Of great interest was the lack of functional expression that we observed with the P2X6 receptor, an effect that appears to be explained by the inability of the P2X6 subunits to form homo-oligomeric assemblies as demonstrated by the lack of co-precipitation of P2X6·FLAG with P2X6·HA. Our findings are in contrast to the original report describing this subunit cDNA, in which HEK 293 cells were also used (12), but are in agreement with findings from two other laboratories that have examined expression using Xenopus oocytes (19, 20). This lack of assembly and function in our hands suggests the possibility that subtle differences in cell culturing (e.g., source of cells, passage number of cells used at time of transfection, or differences in media/sera) can influence other cellular factors that are involved in the assembly of this subunit into homo- but not hetero-oligomers. Such a factor(s) may provide an important target(s) for regulation of functional P2X receptor formation in different cell types and/or tissues.

In situ hybridization studies suggest that different P2X receptor subunits are not only co-expressed in the same tissues but also may co-localize to the same cells (10, 12, 13). These findings raise the possibility that P2X receptor subunits might form hetero-oligomeric channel assemblies. In the present study, we have utilized a co-immunoprecipitation assay to examine whether the seven P2X receptor subunits can associate with one another, and if so, which combinations are productive in forming hetero-oligomeric assemblies. Our results demonstrate that hetero-oligomeric assembly can occur and that it does so in a preferential fashion. We provide direct biochemical evidence for the specific and detergent-stable association of a number of P2X subunit combinations, which are shown in Table I. In contrast, under similar conditions, we did not detect protein-protein interactions for P2X1/P2X4, P2X2/P2X4, P2X3/P2X4, or P2X3/P2X6, or for any combination containing P2X7 subunits. These findings of specific subunit-subunit interactions support the validity of using co-immunoprecipitations as a means of characterizing the co-assembly attributes of P2X subunits. The fact that P2X6 can co-assemble into hetero-oligomeric assemblies with many other subunits but not with itself suggests the possibility that P2X6 subunits might act in a regulatory role within hetero-oligomeric assemblies, similar to what has been shown for the beta  and gamma  subunits of the epithelial sodium channel (26, 27).

                              
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Table I
Summary of observed P2X subunit co-assembly as determined using the co-immunoprecipitation protocols described under "Experimental Procedures"

Whereas most P2X receptor subunits associate with each other to form stable complexes, the P2X7 subunit failed to co-immunoprecipitate with any of the other P2X subunits. This lack of association does not seem to involve the long carboxyl terminus of P2X7 because a truncated form of this receptor subunit did not co-immunoprecipitate the P2X1·HA, P2X2·HA, or P2X3·HA subunits. These results suggest that P2X7 subunits preferentially form homo-oligomeric rather than hetero-oligomeric assemblies. Two recent studies of native preparations support our findings: a report by Tenneti et al. (28) identified two populations of ATP receptors in parotid acinar cells, the properties of which correlated well with those for homo-oligomeric P2X4 and P2X7, and Cario-Toumaniantz et al. (29) provide data suggesting that P2X1 and P2X7 receptors are expressed as separate homo-oligomeric populations in smooth muscle cells.

Expansion from the biochemical experiments to functional studies is needed in order to determine the physiological significance of P2X subunit co-assembly. There are reports describing native receptors that do not match up well with the individual subunits expressed recombinantly, suggesting that hetero-oligomeric assembly might result in the generation of a novel phenotype. The first such study examining P2X co-assembly was for P2X2 and P2X3, which were shown to form a novel hetero-oligomeric channel when co-expressed in HEK 293 cells (10). This phenotype correlated well with the receptors described in some sensory neurons where the two proteins are co-expressed, suggesting that this process occurred in the native setting as well. Additional evidence for hetero-oligomeric assembly has appeared during the course of this study: Le et al. (19) provided functional evidence for a novel channel formed between epitope-tagged P2X4 and P2X6 subunits, and we reported that the co-expression of P2X1 and P2X5 subunits also resulted in a channel with a novel phenotype (22). For both pairs of subunits, there is a marked overlapping pattern of tissue distribution, suggesting that such combinations might be representative of native receptors (12). Therefore, the data presented in this study provide a framework for expansion into the investigation of both recombinant and native hetero-oligomeric P2X receptor phenotypes.

It cannot be presumed a priori that co-expression of two subunits will result in the generation of a novel receptor phenotype. This caveat is borne out when results from this study are taken in conjunction with those by Lewis et al. (10). In that study of P2X subunit co-expression, it was reported that co-transfection of P2X1/P2X2, P2X1/P2X4, and P2X3/P2X4 did not lead to the formation of receptors with novel phenotypes, a finding that was interpreted to mean that the subunits did not co-assemble. Although our results also provide evidence that P2X1/P2X4, and P2X3/P2X4 do not co-assemble, we found that the subunit pair P2X1/P2X2 did co-assemble. This finding, when taken together with the biophysical findings (10), suggests that lack of a novel phenotype does not necessarily mean that two subunits are not interacting to form a hetero-oligomeric channel complex. An alternative explanation is that when two receptor subunits participate to form a complex, one subunit type can dominate the phenotype of the resulting channel, and thus only a single phenotype is observed. This phenomenon is readily observed in the ionotropic glutamate receptor family, where the presence of the GluR-B subunit dominates hetero-oligomeric receptor assemblies with regard to both rectification and calcium permeability properties (30). Taking this point further, if it is the ratio of subunits in an assembly that determines the observed phenotype, and a mixed population of hetero-oligomeric receptor channels containing different ratios of the two subunits form after a co-transfection, then it is possible that two apparent populations of channels, resembling the homo-oligomeric parent receptors, would be detected. This is indeed what was observed by Lewis et al. (10) for the P2X1/P2X2 combination. Even if there is no overt effect on the biophysical phenotype of a receptor, hetero-oligomeric assembly could still have important impact on receptor function; e.g., by altering the responsiveness of a complex to intracellular regulation or its domain targeting within a cell.

As an attempt to understand how hetero-oligomeric assembly alters the functional properties of these channels, we are now investigating the functional consequences of the specific interactions derived from this study. Our biochemical data provide specific templates to start elucidating the subunit composition of endogenous P2X receptors. In addition, the specificity of the interactions among the members of the P2X receptor family should provide a useful tool in investigating the molecular determinants involved in hetero-oligomeric P2X receptor formation, results that could have applications beyond just these ATP-gated channels.

    FOOTNOTES

* This work was supported by National Institute of Health Grants NS35534 (to M. M. V.) and HL56236 (to T. M. E.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence and reprint requests should be addressed: Dept. of Pharmacological and Physiological Science, Saint Louis University School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104. Tel.: 314-577-8545; Fax: 314-577-8233; E-mail: voigtm{at}slu.edu.

2 G. E. Torres, T. M. Egan, and M. M. Voigt, unpublished observation.

    ABBREVIATIONS

The abbreviations used are: HA, hemagglutinin; HEK 293, human embryonic kidney 293.

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Abstract
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