P2X 4 Receptor Is a Glycosylated Cardiac Receptor Mediating a Positive Inotropic Response to ATP*

, Although P2X receptors are suggested to play a role in synaptic neurotransmission, the specific physiological role of each P2X receptor subtype remains largely un-known. We used cultured chick embryo ventricular myocytes as a model to study a potential physiological role of the P2X 4 receptor in mediating the positive ino- tropic effect of ATP. The chick P2X4 receptor (cP2X 4 R) mRNA was expressed in the heart and the pharmacological features of the ATP-induced positive inotropic response were similar to those of the cP2X 4 R in terms of insensitivity to blockade by known P2 receptor antago-nists and the ineffectiveness of adenosine 5 (cid:1) -( (cid:1) , (cid:2) -meth-ylene)triphosphate as an agonist. Treatment of myocytes with antisense oligonucleotides specific to the 5 (cid:1) region of cP2X 4 R abrogated the P2 agonist-stimulated 45 Ca influx. Similarly, antisense oligonucleotide treatment also blocked the 2-methylthio-ATP-stimulated increase in contractile amplitude. The data suggest that the native P2X 4 receptor is involved in mediating the P2 agonist-stimulated response in the heart. In character-izing the biochemical property of the P2X 4 receptor, antibody against cP2X

P2X receptors are nonselective cation channels gated by ATP (for reviews, see Refs. [1][2][3]. Seven isoforms of P2X receptors identified so far show 35-50% sequence identities based on comparison of the homologous region. Each receptor has two transmembrane domains and a large extracellular loop that contains a number of putative N-glycosylation sites and an ATP binding site. Recent human genome data suggested three additional possible P2X receptors in the human genome, and no P2X counterparts were found in the yeast or Drosophila genome (4). Both N-and C-intracellular termini have been shown to be important in determining the rate of desensitization of the P2X receptor (1)(2)(3). Biochemical and elctrophysiological studies suggested homomeric and heteromeric structures, possibly trimer, as the functional form of P2X receptors (1)(2)(3)(5)(6)(7).
In vitro and in vivo studies indicated that P2X receptors are involved in a wide range of functions in synaptic transmission and in excitation of smooth muscle and inflammatory cells (1)(2)(3). Various phenotypes were found in P2X-deficient mice, such as reduced fertility in P2X 1 R knockout male mice (8) and reduced sensitivity to pain in P2X 3 R knockout mice (9). However, the physiological function of individual P2X receptors remains largely unknown.
The P2X 4 receptor was chosen for study of its physiological role for the following reasons. First, the pharmacological features of the native P2 receptor-mediated increase in cardiac myocyte contractility were most similar to those of the P2X 4 receptor. Thus, the P2 agonist 2-methylthio-ATP (2-meSATP) 1 is an efficacious agonist at stimulating both the myocyte contractility and the P2X 4 receptor-mediated current, whereas AMP-CPP is a weak agonist (18). Further, the increase in myocyte contractility was insensitive to blockade by the P2 antagonist suramin or pyridoxal phosphate-6-azophenyl-2Ј,4Јdisulfonic acid, as was the P2X 4 receptor-mediated increase in current. Finally, the receptor was expressed in the heart, and its cardiac transgenic overexpression resulted in an increased basal contractility of the intact heart, mediated via the endogenous ATP stimulating the overexpressed P2X 4 receptor (11).
Most membrane receptors are glycoproteins. The oligosacchrides are involved in various biological activities including intracellular trafficking, maintenance of receptor stability, recognition of ligand, and proper folding of the receptor. N-Linked glycosylation of the thrombin receptor in T lymphoid cells or of the insulin-like growth factor-1 receptor in Ewing's sarcoma cells was shown to be important for localization of the receptor on plasma membrane (12,13). However, glycosylation of the human parathyroid hormone/parathyroid hormone-related protein receptor was not important for their expression at the cell surface or for the integrity of their functional responsiveness (14). N-Linked glycosylation of the rat P2X 2 and P2X 1 * This work was supported by an Established Investigatorship Award from the American Heart Association and by National Institutes of Health RO1 Grant HL48225 (to B. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  receptors was important for their cell surface expression (15)(16)(17). Because the P2X 4 receptor is a cell surface ligand-gated ion channel, studying the biochemical determinant of the receptor's localization on plasma membrane is highly relevant to its physiological function. Thus, another objective of the study was to test the possible glycosylation of P2X 4 receptor and to study the role of such glycosylation in the cell surface expression of the P2X 4 receptor.

DNA Constructs and Preparation of Sense and Antisense
Oligonucleotides-Chick P2X 4 R full-length clone was isolated from a 17-day-old embryo brain cDNA library (custom made; Stratagene) using as a probe a partial cP2X 4 sequence obtained by degenerate PCR on RNA isolated from cultured cardiac ventricular myocytes as previously described (10) and subcloned into the pcDNA3 expression vector. The full-length cDNA was then subcloned in pcDNA3 expression vector as previously described (10). Phosphorothioated sense (CCGGCCGCCGCCATGGCT) and antisense (AGCCATGGCGGCGGCCGG) oligonucleotides overlapping the 5Ј-untranslated region and translation start region of chick P2X 4 cDNA were synthesized by the Cell Center at the University of Pennsylvania (start code location underlined).
Preparation of Cultured Cardiac Cells, Transfection with cP2X 4 Receptor cDNA, and Measurement of Contractile Amplitude and of 45 Ca Uptake-Cardiac ventricular cells were cultured from chick embryos 14 days in ovo according to previously described procedures (18). Cells were maintained in cultures for 12 h before being exposed to the calcium phosphate/DNA precipitates for 6 h at 37°C (19). After two washes with fresh growth medium, the myocytes were cultured for an additional 24 or 36 h as indicated under "Results." For treatment with sense and antisense oligonucleotides, 1 M sense or antisense oligonucleotides were included in calcium phosphate transfection mixtures. After 6 h, the medium was replaced with that containing the oligonucleotides for additional 2 days, at which time the effect on cell contraction and 45 Ca uptake were measured. For inhibition of N-linked glycosylation, the cells transfected with the cP2X 4 R cDNA 12 h earlier were washed twice with fresh growth medium, and the cells were then maintained in the culture medium containing 1 g/ml tunicamycin (Sigma) for an additional 24 -36 h. Myocyte cultures were incubated with sense or antisense oligonucleotides as described above. The effect of 2-meSATP-stimulated 45 Ca uptake was measured according to a previously described method (18).
Immunobotting and RNA Blotting-Chick P2X 4 antiserum was raised in rabbit against a peptide (KKYKYVEDYELGTSET) corresponding to the C terminus of chick P2X 4 receptor (Cocalico Biologicals, Inc., Reamstown, PA). Isolated cardiac cells were solubilized in SDS-PAGE sample buffer, and 50 g of solubilized proteins were resolved by 10% SDS-PAGE and transferred to nitrocellulose membrane, followed by probing with the rabbit polyclonal antibody. Following several washes, the nitrocellulose membrane was incubated with peroxidasecoupled anti-rabbit Ig antibody (1:5000) and developed with an ECL-Plus kit (Amersham Biosciences). The same antibody incubated with its peptide antigen was used as a negative control.
Poly(A) ϩ mRNAs were isolated from tissues obtained from 14-day-old chicken embryos using the FastTrack kit 2.0 (Invitrogen). Ten micrograms of mRNA per tissue were separated in a 1% agarose gel containing formaldehyde, capillary-transferred onto a positively charged nylon membrane (Roche Molecular Biochemicals), and UV-cross-linked. The membrane was hybridized with a [␣-32 P]dCTP-labeled probe containing the complete coding region of cP2X 4 . The hybridization was performed in ExpressHyb solution (CLONTECH) at 68°C for 1 h, and the final wash of the membrane was done in 0.1ϫ SSC, 0.1ϫ SDS at 50°C. The membrane was exposed to x-ray film for 3 days.
Biotinylation and Cross-linking of the Cell Surface P2X 4 Receptor-Chick cardiac cells were washed three times with serum-free culture medium, labeled by N-hydroxysuccinimide-SS-biotin (Pierce; 1 mg/ml in serum-free culture medium) for 20 min in the CO 2 incubator. The labeled cells were washed three times with the same medium and extracted by SDS extraction buffer (40 mM Tris-Cl, pH 8.0, 1.3% SDS, 10 l/ml protease inhibitor mixture from Sigma). Myocyte extracts were diluted to 0.5% SDS by TET buffer (100 mM Tris-Cl, pH 8.0, 10 mM EDTA, and 1% Triton X-114, 10 l/ml protease inhibitor mixture), and the biotinylated cell surface proteins were precipitated by streptavidinconjugated agarose (Pierce) in the same buffer. The precipitated proteins were subjected to SDS-PAGE and probed in Western blot by antiserum against chick P2X 4 receptor (1:600 dilution).

RESULTS AND DISCUSSION
P2X 4 Receptor Is a Native Cardiac Receptor-The physiological role of the recently identified ligand-gated P2X receptors is an area of much investigative interest. Whereas some P2X receptor-null mice suggested an important role of the P2X 1 receptor in male fertility and of the P2X 3 receptor in coding the intensity of warm stimuli (8,9), the biological role of other P2X receptor subtypes remained unclear. In the present study, a physiological role of the P2X 4 receptor was investigated. Cultured chick cardiac cells were chosen as a model for the following reasons. First, the chick P2X 4 receptor (cP2X 4 R) mRNA was expressed in the chick heart. Fig. 1 showed that the cP2X 4 R transcript in the heart was expressed at the same or a higher level than in the brain, although lungs have the highest level among the three tissues. In fact, the chick P2X 4 receptor cDNA was cloned based on degenerate PCR on RNA isolated from the cultured chick cardiac cell (10). Second, in addition to being expressed in the heart, the pharmacological features of chick P2X 4 R-mediated ionic current also appeared similar to those of the native P2 receptor that mediated the ATP-induced increase in myocyte contractility (10,18). Thus, 2-meSATP was a more efficacious agonist than AMP-CPP at the cloned cP2X 4 receptor and at stimulating the native chick P2 receptor-mediated increase in myocyte contractility (18). The P2 receptor antagonist suramin (50 M) did not block the native P2 receptor-mediated increase in myocyte contractile amplitude by 2-meSATP. The percentage of stimulation above basal level in response to 2-meSTAP was 58 Ϯ 15%, similar to the 53 Ϯ 20% increase when suramin was also present (n ϭ 8 cells from 4 cultures, p Ͼ 0.1 test) (Fig. 2). Similarly, the P2 antagonist pyridoxal phosphate-6-azophenyl-2Ј,4Ј-disulfonic acid (50 M) did not block the 2-meSATP-stimulated increase in myocyte contractility (data not shown). Although more work is needed, the pharmacological features of the P2 agonist-induced contractile response of the chick embryo cardiac myocyte are similar to those of the response in adult rat and mouse cardiac myocytes (11,18). Since the P2X 4 receptor was also detected in these adult mammalian cardiac myocytes, the present data are consistent with the idea that the chick cardiac cell is a potentially good model to investigate the physiological role and biochemical property of the cardiac P2X 4 R.
Finally, the cultured chick cardiac cells were amenable to the gene or cDNA transduction approach, which complemented functional (such as calcium influx and contractility) and biochemical (such as cell surface receptor expression) studies. This is an important advantage because of the feasibility in comparing the biochemical and the functional data in the same cell model.
To more definitively determine the function of the P2X 4 receptor in mediating the 2-meSATP-stimulated increase in myocyte contractility, antisense oligonucleotide directed at the 5Ј-untranslated region contiguous with the translation start site of the cP2X 4 R was prepared. The antisense oligonucleotide against the cP2X 4 R effectively blocked the expression of the exogenous cP2X 4 R when both the oligonucleotide and the cP2X 4 R cDNA were transduced into the myocyte (Fig. 3a). The anti-cP2X 4 R antibody was unable to detect the native cP2X 4 R due to a low level expression of the native P2X 4 R or a low affinity of the antibody for the receptor or both. Therefore, antisense treatment of the myocytes expressing only native P2X 4 R could not have detected any decrease in its level using this antibody. On the other hand, overexpression of the exogenous recombinant receptor would allow the antibody to detect the receptor and thus the effect of antisense oligonucleotide treatment on the level of the receptor. That the antisense treatment effectively decreased the level of the overexpressed recombinant cP2X 4 R suggested that the same treatment should also have reduced or eliminated the native P2X 4 receptor. Use of the cP2X 4 R-overexpressing myocyte was to demonstrate that antisense treatment can effectively reduce the level of the receptor.
If the native cP2X 4 R is involved in mediating the 2-meSATPstimulated response, one would expect that such antisense oligonucleotide treatment would inhibit such a response. The present data demonstrated that this was indeed the case. Antisense oligonucleotide treatment abrogated the P2 agoniststimulated 45 Ca influx (Ϫ17 Ϯ Ϫ7.8%, n ϭ 3, Ϯ S.E.) (Fig. 3, b  and c). Similarly, antisense oligonucleotide treatment also blocked the 2-meSATP-stimulated increase in contractile amplitude (3.1 Ϯ 1%, n ϭ 13 cells from four cultures) (Fig. 3, b and  d). On the other hand, myocytes transfected with pcDNA3 (mock-transfected) or with the sense oligonucleotide showed increases of 41.6 Ϯ 13.8 and 61 Ϯ 20% (n ϭ 3, Ϯ S.E.) in the level of 45 Ca influx in response to 2-meSATP, respectively. Sense oligonucleotide-treated myocytes showed a preserved response to 2-meSATP with a 33 Ϯ 3.3% (n ϭ 10 cells from four cultures) increase in contractile amplitude (Fig. 3, b and d).
Overall, these data provide more definitive evidence that the P2X 4 receptor serves a physiological role, that of mediating an increase in the myocyte contractility and calcium influx in response to ATP.
P2X 4 Receptor Is a Glycosylated Protein-Having established an important physiological role for the P2X 4 R, its further bio-chemical characterization was carried out. The cP2X 4 R detected on the Western blot represented the exogenous recombinant chick P2X 4 R that was overexpressed following transfection of its cDNA. The anti-cP2X 4 R antibody detected two major bands, 58-and 44 -45-kDa proteins, in an immunoblot of membranes from cP2X 4 R-overexpressing myocytes (Fig.  3A). The detected molecular mass of the P2X 4 receptor in immunoblotting was 58 kDa, consistent with that described by others for the rat P2X 4 receptor (20,21). Because the calculated molecular mass of P2X 4 receptor is 44 -45 kDa, the greater detected molecular mass of 58 kDa may be due to a posttranslational modification such as glycosylation, given the presence of seven potential glycosylation sites in the extracellular loop of the P2X 4 receptor (16). To test this possibility, the P2X 4 R-overexpressing myocytes were treated with tunicamycin, a widely used specific inhibitor of N-linked glycosylation. Tunicamycin caused the disappearance of the 58-kDa band (Fig. 4a). A similar result was obtained when myocytes overexpressing the human P2X 4 R were treated with tunicamycin (data not shown). These data supported the notion that the  1, 2, and 3, respectively). The solubilized extracts (in SDS sample buffer) were run on a SDS-PAGE and blotted with antibody against cP2X 4 R. Data were representative of three experiments. P2X 4 receptor is glycosylated in cardiac myocytes, similar to the glycosylation of P2X 1 and P2X 2 receptors (15,16). At later time points following the transfection with exogenous cP2X 4 R cDNA, only the 58-kDa receptor protein was present (Fig. 4b), consistent with a more complete glycosylation of the exogenous cP2X 4 R. Only the 58-kDa P2X 4 receptor was detectable in cardiac myocytes isolated from adult wild type mouse and from P2X 4 receptor transgenic mice, which showed an increased receptor-mediated contractility (11). These data suggested that the 58-kDa receptor protein, probably representing the glycosylated P2X 4 receptor, is the final and functional form of the receptor. At the intermediate time point (i.e. 36 -48 h following transfection with the cP2X 4 R cDNA), the presence of both glycosylated and nonglycosylated forms of the receptor provided a unique system to further study the role of glycosylation in maintaining the biochemical property of P2X 4 receptors.
Glycosylation Is Required for Cell Surface Expression of the P2X 4 Receptor and for Maintaining Its Detergent Solubility-To characterize the role of N-linked glycosylation in modulating the biochemical property of the P2X 4 receptor, studies were carried out to test the hypothesis that glycosylation is needed for cell surface expression of the P2X 4 receptor and for maintaining the detergent solubility of the receptor. Glycosyl-ation has been reported to play an important role in intracellular trafficking and in the cell surface localization of various receptors and many other membrane-associated proteins. To test this possibility on the P2X 4 receptor, cP2X 4 R-overexpressing myocytes were biotinylated to label the cell surface receptor and treated with or without tunicamycin. The labeled receptor was extracted and precipitated by streptavidin-agarose beads. Immunoblotting with the anti-cP2X 4 R antibody showed that tunicamycin could completely block the cell surface expression of P2X 4 receptor (Fig. 5b), suggesting that N-linked glycosylation is required for its localization on the plasma membrane.
While SDS sample buffer could extract both glycosylated and nonglycosylated P2X 4 receptors, only the glycosylated receptor can be solubilized by 1% Triton X-114 (Fig. 4a). In fact, the nonglycosylated receptor (the 44 -45-kDa protein) was resistant to various extraction conditions such as 1% Triton X-114 in the presence or the absence of 1% deoxycholic acid or 0.5 M urea (Fig. 6) or other conditions such as 1% Nonidet P-40 or 1 M urea (not shown). SDS, at the reduced concentration of 1.3%, was able to solubilize the 44 -45-kDa nonglycosylated receptor (Fig.  5a). However, only the 58-kDa receptor, and not the 44 -45-kDa receptor (also extracted by 1.3% SDS), could be precipitated by streptavidin (Fig. 5b). Thus, the 44 -45-kDa receptor was not labeled with biotin, indicating that the nonglycosylated P2X 4 receptor failed to localize on the plasma membrane. Together, these data suggested that glycosylation was necessary for maintaining the receptor's detergent solubility property and cell surface expression. Showing that the cell surface localization of the receptor requires glycosylation is clearly important for the function of these receptors, since they are ligand-gated receptor channels on the plasma membrane. Overall, the present study revealed a novel physiological function of the P2X 4 receptor and indicated the importance of N-linked glycosylation in maintaining its biochemical property and cell surface expression.
Acknowledgments-We are grateful to Dr. Anja Ruppelt for experimental help and to Dr. Walter Stü hmer for generous support.
FIG. 5. Effect of tunicamycin treatment on the cell surface localization of P2X 4 receptor. a, cells transfected with cP2X 4 R cDNA were treated with or without tunicamycin and then subjected to extraction with 1.3% SDS to solubilize the glycosylated and nonglycosylated cP2X 4 R as described in the legend to Fig. 4a. After centrifugation at 12,000 ϫ g for 15 min, the supernatant was further separated on a SDS-PAGE and blotted with cP2X 4 R antibody. Data were typical of three other experiments. b, cardiac ventricular cells were prepared, transfected with cP2X 4 R cDNA, and treated with tunicamycin or not treated. Cells were then subjected to cross-linking with N-hydroxysuccinimide-SS-biotin. Following extraction with 1.3% SDS for 20 min, the supernatant was obtained after centrifugation at 12,000 ϫ g for 15 min. The cP2X 4 R in the supernatant was precipitated by streptavidin-conjugated agarose beads and detected by blotting with the antibody against cP2X 4 R. Data are representative of three other experiments.
FIG. 6. Ability of various detergents to solubilize the glycosylated and nonglycosylated cP2X 4 receptor. Cardiac ventricular cells were prepared, transfected with cP2X 4 R cDNA, and subjected to cross-linking with N-hydroxysuccinimide-SS-biotin as described under "Experimental Procedures." Cells were then subjected to treatment with the various detergent combinations using the method described in the legend to Fig. 5. Part of the supernatant (Supernatant), obtained following the detergent solubilization, and the pellet fraction (Pellet) were dissolved in SDS-PAGE sample buffer and blotted with antibody against cP2X 4 R. Another portion of the supernatant was precipitated with streptavidin-conjugated agarose beads (IP), solubilized in SDS-PAGE, and blotted with antibody against cP2X 4 R. Data are representative of three other experiments.