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P2X4 Receptor Is a Glycosylated Cardiac Receptor Mediating a Positive Inotropic Response to ATP

2002; Elsevier BV; Volume: 277; Issue: 18 Linguagem: Inglês

10.1074/jbc.m112097200

1083-351X

Bing Hu, Carol Senkler, Alexander Yang, Florentina Soto, Bruce T. Liang,

Genetics and Neurodevelopmental Disorders

Although P2X receptors are suggested to play a role in synaptic neurotransmission, the specific physiological role of each P2X receptor subtype remains largely unknown. We used cultured chick embryo ventricular myocytes as a model to study a potential physiological role of the P2X4 receptor in mediating the positive inotropic effect of ATP. The chick P2X4 receptor (cP2X4R) mRNA was expressed in the heart and the pharmacological features of the ATP-induced positive inotropic response were similar to those of the cP2X4R in terms of insensitivity to blockade by known P2 receptor antagonists and the ineffectiveness of adenosine 5′-(α,β-methylene)triphosphate as an agonist. Treatment of myocytes with antisense oligonucleotides specific to the 5′ region of cP2X4R abrogated the P2 agonist-stimulated 45Ca influx. Similarly, antisense oligonucleotide treatment also blocked the 2-methylthio-ATP-stimulated increase in contractile amplitude. The data suggest that the native P2X4 receptor is involved in mediating the P2 agonist-stimulated response in the heart. In characterizing the biochemical property of the P2X4 receptor, antibody against cP2X4R detected a 44-kDa and a 58-kDa protein in the immunoblot. Inhibition of N-linked glycosylation by tunicamycin converted the 58-kDa protein to the 44-kDa protein, suggesting that the 58-kDa protein was a glycosylated P2X4receptor. The nonglycosylated 44-kDa P2X4 receptor was resistant to various detergent/aqueous extraction, consistent with a role of glycosylation in maintaining its detergent solubility and hydrophilicity. Cross-linking the cell surface proteins withN-hydroxysuccinimide-SS-biotin followed by affinity precipitation with streptavidin-conjugated agarose and subsequent immunoblotting with anti-cP2X4R showed that only the glycosylated 58-kDa P2X4 receptor was expressed on the cell surface, indicating an important role of glycosylation for the receptor’s localization on the plasma membrane. These data revealed a novel physiologic function of the P2X4 receptor and suggested the importance of N-linked glycosylation in its cell surface expression and detergent solubility. Although P2X receptors are suggested to play a role in synaptic neurotransmission, the specific physiological role of each P2X receptor subtype remains largely unknown. We used cultured chick embryo ventricular myocytes as a model to study a potential physiological role of the P2X4 receptor in mediating the positive inotropic effect of ATP. The chick P2X4 receptor (cP2X4R) mRNA was expressed in the heart and the pharmacological features of the ATP-induced positive inotropic response were similar to those of the cP2X4R in terms of insensitivity to blockade by known P2 receptor antagonists and the ineffectiveness of adenosine 5′-(α,β-methylene)triphosphate as an agonist. Treatment of myocytes with antisense oligonucleotides specific to the 5′ region of cP2X4R abrogated the P2 agonist-stimulated 45Ca influx. Similarly, antisense oligonucleotide treatment also blocked the 2-methylthio-ATP-stimulated increase in contractile amplitude. The data suggest that the native P2X4 receptor is involved in mediating the P2 agonist-stimulated response in the heart. In characterizing the biochemical property of the P2X4 receptor, antibody against cP2X4R detected a 44-kDa and a 58-kDa protein in the immunoblot. Inhibition of N-linked glycosylation by tunicamycin converted the 58-kDa protein to the 44-kDa protein, suggesting that the 58-kDa protein was a glycosylated P2X4receptor. The nonglycosylated 44-kDa P2X4 receptor was resistant to various detergent/aqueous extraction, consistent with a role of glycosylation in maintaining its detergent solubility and hydrophilicity. Cross-linking the cell surface proteins withN-hydroxysuccinimide-SS-biotin followed by affinity precipitation with streptavidin-conjugated agarose and subsequent immunoblotting with anti-cP2X4R showed that only the glycosylated 58-kDa P2X4 receptor was expressed on the cell surface, indicating an important role of glycosylation for the receptor’s localization on the plasma membrane. These data revealed a novel physiologic function of the P2X4 receptor and suggested the importance of N-linked glycosylation in its cell surface expression and detergent solubility. 2-methylthio-ATP adenosine 5′-(α,β-methylene)triphosphate P2X receptors are nonselective cation channels gated by ATP (for reviews, see Refs. 1Khakh B.S. Burnstock G. Kennedy C. King B.F. North R.A. Seguela P. Voigt M. Humphrey P.A. Pharmacol. Rev. 2001; 53: 107-118PubMed Google Scholar, 2Soto F. Garcia-Guzman M. Stuhmer W. J. Membr. Biol. 1997; 160: 91-100Crossref PubMed Scopus (83) Google Scholar, 3Ralevic V. Burnstock G. Pharmacol. Rev. 1998; 50: 413-492PubMed Google Scholar). 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 putativeN-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 orDrosophila genome (4Venter J.C. Adams M.D. Li E.W. Myers P.W. Mural R.J. Sutton G.G. Smith H.O. Yandell M. Evans C.A. Holt R.A. Gocayne J.D. Amanatides P. Ballew R.M. Huson D.H. Wortman J.R. et al.Science. 2001; 291: 1304-1351Crossref PubMed Scopus (10634) Google Scholar). Both N- and C-intracellular termini have been shown to be important in determining the rate of desensitization of the P2X receptor (1Khakh B.S. Burnstock G. Kennedy C. King B.F. North R.A. Seguela P. Voigt M. Humphrey P.A. Pharmacol. Rev. 2001; 53: 107-118PubMed Google Scholar, 2Soto F. Garcia-Guzman M. Stuhmer W. J. Membr. Biol. 1997; 160: 91-100Crossref PubMed Scopus (83) Google Scholar, 3Ralevic V. Burnstock G. Pharmacol. Rev. 1998; 50: 413-492PubMed Google Scholar). Biochemical and elctrophysiological studies suggested homomeric and heteromeric structures, possibly trimer, as the functional form of P2X receptors (1Khakh B.S. Burnstock G. Kennedy C. King B.F. North R.A. Seguela P. Voigt M. Humphrey P.A. Pharmacol. Rev. 2001; 53: 107-118PubMed Google Scholar, 2Soto F. Garcia-Guzman M. Stuhmer W. J. Membr. Biol. 1997; 160: 91-100Crossref PubMed Scopus (83) Google Scholar, 3Ralevic V. Burnstock G. Pharmacol. Rev. 1998; 50: 413-492PubMed Google Scholar, 5Kim M. Yoo O.J. Choe S. Biochem. Biophys. Res. Commun. 1997; 240: 618-622Crossref PubMed Scopus (61) Google Scholar, 6Nicke A. Baumert H.G. Rettinger J. Eichele A. Lambrecht G. Mutschler E. Schmalzing G. EMBO J. 1998; 17: 3016-3028Crossref PubMed Scopus (483) Google Scholar, 7Stoop R. Thomas S. Rassendren F. Kawashima E. Buell G. Surprenant A North R. Mol. Pharmacol. 1999; 56: 973-981Crossref PubMed Scopus (117) Google Scholar). 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 (1Khakh B.S. Burnstock G. Kennedy C. King B.F. North R.A. Seguela P. Voigt M. Humphrey P.A. Pharmacol. Rev. 2001; 53: 107-118PubMed Google Scholar, 2Soto F. Garcia-Guzman M. Stuhmer W. J. Membr. Biol. 1997; 160: 91-100Crossref PubMed Scopus (83) Google Scholar, 3Ralevic V. Burnstock G. Pharmacol. Rev. 1998; 50: 413-492PubMed Google Scholar). Various phenotypes were found in P2X-deficient mice, such as reduced fertility in P2X1R knockout male mice (8Mulryan K. Gitterman D.P. Lewis C.J. Vial C. Leckie B.J. Cobb A.L. Brown J.E. Conley E.C. Buell G. Pritchard C.A. Evans R.J. Nature. 2000; 403: 86-89Crossref PubMed Scopus (335) Google Scholar) and reduced sensitivity to pain in P2X3R knockout mice (9Souslova V. Cesare P. Ding Y. Akoplan A.N. Stanfa L. Suzuki R. Carpenter K. Dickenson A. Boyce S. Hill R. Nebenius-Oosthuizen D. Smith A.J.H. Kidd E.J. Wood J.N. Nature. 2000; 407: 1015-1017Crossref PubMed Scopus (392) Google Scholar). However, the physiological function of individual P2X receptors remains largely unknown. The P2X4 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 P2X4receptor. Thus, the P2 agonist 2-methylthio-ATP (2-meSATP)1 is an efficacious agonist at stimulating both the myocyte contractility and the P2X4 receptor-mediated current, whereas AMP-CPP is a weak agonist (18Xu E. Podrasky D. Liang B.T. Am. J. Physiol. 1997; 273: H2380-H2387PubMed Google Scholar). Further, the increase in myocyte contractility was insensitive to blockade by the P2 antagonist suramin or pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid, as was the P2X4 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 P2X4 receptor (11Hu B. Mei Q. Smith E. Barry W.H. Liang B.T. FASEB J. 2001; 10: 2739-2741Google Scholar). 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 (12Tordai A. Brass L.F. Gelfand E.W. Biochem. Biophys. Res. Commun. 1995; 206: 857-862Crossref PubMed Scopus (27) Google Scholar, 13Girnita L. Wang M. Xie Y. Nilsson G. Dricu A. Wejde J. Larsson O. Anticancer Drug Des. 2000; 15: 67-72PubMed Google Scholar). 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 (14Bisello A. Greenberg Z. Behar V. Rosenblatt M. Suva I.J. Biochemistry. 1996; 35: 15890-15895Crossref PubMed Scopus (57) Google Scholar). N-Linked glycosylation of the rat P2X2 and P2X1 receptors was important for their cell surface expression (15Torres G.E. Egan T.M. Voigt M.M. Biochemistry. 1998; 37: 14845-14851Crossref PubMed Scopus (58) Google Scholar, 16Rettinger J. Aschrafi A. Schmalzing G. J. Biol. Chem. 2000; 275: 33542-33547Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 17Newbolt A Stoop R. Virginio C. Surprenant A. North R.A. Buell G. Rassendren F. J. Biol. Chem. 1998; 273: 15177-15182Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Because the P2X4 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 P2X4receptor and to study the role of such glycosylation in the cell surface expression of the P2X4 receptor. Chick P2X4R full-length clone was isolated from a 17-day-old embryo brain cDNA library (custom made; Stratagene) using as a probe a partial cP2X4 sequence obtained by degenerate PCR on RNA isolated from cultured cardiac ventricular myocytes as previously described (10Ruppelt A. Liang B.T. Soto S. Prog. Brain Res. 1999; 120: 81-90Crossref PubMed Google Scholar) and subcloned into the pcDNA3 expression vector. The full-length cDNA was then subcloned in pcDNA3 expression vector as previously described (10Ruppelt A. Liang B.T. Soto S. Prog. Brain Res. 1999; 120: 81-90Crossref PubMed Google Scholar). Phosphorothioated sense (CCGGCCGCCGCCATGGCT) and antisense (AGCCATGGCGGCGGCCGG) oligonucleotides overlapping the 5′-untranslated region and translation start region of chick P2X4 cDNA were synthesized by the Cell Center at the University of Pennsylvania (start code location underlined). Cardiac ventricular cells were cultured from chick embryos 14 days in ovoaccording to previously described procedures (18Xu E. Podrasky D. Liang B.T. Am. J. Physiol. 1997; 273: H2380-H2387PubMed Google Scholar). Cells were maintained in cultures for 12 h before being exposed to the calcium phosphate/DNA precipitates for 6 h at 37 °C (19Xu H Miller J. Liang B.T. Nucleic Acids Res. 1992; 20: 6425-6426Crossref PubMed Scopus (25) Google Scholar). 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 μmsense 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 45Ca uptake were measured. For inhibition of N-linked glycosylation, the cells transfected with the cP2X4R cDNA 12 h earlier were washed twice with fresh growth medium, and the cells were then maintained in the culture medium containing 1 μg/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 45Ca uptake was measured according to a previously described method (18Xu E. Podrasky D. Liang B.T. Am. J. Physiol. 1997; 273: H2380-H2387PubMed Google Scholar). Chick P2X4antiserum was raised in rabbit against a peptide (KKYKYVEDYELGTSET) corresponding to the C terminus of chick P2X4 receptor (Cocalico Biologicals, Inc., Reamstown, PA). Isolated cardiac cells were solubilized in SDS-PAGE sample buffer, and 50 μg 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 peroxidase-coupled 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)+ 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 [α-32P]dCTP-labeled probe containing the complete coding region of cP2X4. The hybridization was performed in ExpressHyb solution (CLONTECH) at 68 °C for 1 h, and the final wash of the membrane was done in 0.1× SSC, 0.1× SDS at 50 °C. The membrane was exposed to x-ray film for 3 days. Chick cardiac cells were washed three times with serum-free culture medium, labeled byN-hydroxysuccinimide-SS-biotin (Pierce; 1 mg/ml in serum-free culture medium) for 20 min in the CO2incubator. The labeled cells were washed three times with the same medium and extracted by SDS extraction buffer (40 mmTris-Cl, pH 8.0, 1.3% SDS, 10 μl/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 μl/ml protease inhibitor mixture), and the biotinylated cell surface proteins were precipitated by streptavidin-conjugated agarose (Pierce) in the same buffer. The precipitated proteins were subjected to SDS-PAGE and probed in Western blot by antiserum against chick P2X4 receptor (1:600 dilution). 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 P2X1receptor in male fertility and of the P2X3 receptor in coding the intensity of warm stimuli (8Mulryan K. Gitterman D.P. Lewis C.J. Vial C. Leckie B.J. Cobb A.L. Brown J.E. Conley E.C. Buell G. Pritchard C.A. Evans R.J. Nature. 2000; 403: 86-89Crossref PubMed Scopus (335) Google Scholar, 9Souslova V. Cesare P. Ding Y. Akoplan A.N. Stanfa L. Suzuki R. Carpenter K. Dickenson A. Boyce S. Hill R. Nebenius-Oosthuizen D. Smith A.J.H. Kidd E.J. Wood J.N. Nature. 2000; 407: 1015-1017Crossref PubMed Scopus (392) Google Scholar), the biological role of other P2X receptor subtypes remained unclear. In the present study, a physiological role of the P2X4receptor was investigated. Cultured chick cardiac cells were chosen as a model for the following reasons. First, the chick P2X4 receptor (cP2X4R) mRNA was expressed in the chick heart. Fig. 1showed that the cP2X4R 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 P2X4 receptor cDNA was cloned based on degenerate PCR on RNA isolated from the cultured chick cardiac cell (10Ruppelt A. Liang B.T. Soto S. Prog. Brain Res. 1999; 120: 81-90Crossref PubMed Google Scholar). Second, in addition to being expressed in the heart, the pharmacological features of chick P2X4R-mediated ionic current also appeared similar to those of the native P2 receptor that mediated the ATP-induced increase in myocyte contractility (10Ruppelt A. Liang B.T. Soto S. Prog. Brain Res. 1999; 120: 81-90Crossref PubMed Google Scholar, 18Xu E. Podrasky D. Liang B.T. Am. J. Physiol. 1997; 273: H2380-H2387PubMed Google Scholar). Thus, 2-meSATP was a more efficacious agonist than AMP-CPP at the cloned cP2X4 receptor and at stimulating the native chick P2 receptor-mediated increase in myocyte contractility (18Xu E. Podrasky D. Liang B.T. Am. J. Physiol. 1997; 273: H2380-H2387PubMed Google Scholar). The P2 receptor antagonist suramin (50 μm) 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 ± 15%, similar to the 53 ± 20% increase when suramin was also present (n = 8 cells from 4 cultures, p > 0.1 test) (Fig.2). Similarly, the P2 antagonist pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (50 μm) 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 (11Hu B. Mei Q. Smith E. Barry W.H. Liang B.T. FASEB J. 2001; 10: 2739-2741Google Scholar, 18Xu E. Podrasky D. Liang B.T. Am. J. Physiol. 1997; 273: H2380-H2387PubMed Google Scholar). Since the P2X4 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 P2X4R.Figure 2P2 receptor agonist-stimulated increase in myocyte contractility is insensitive to blockade by receptor antagonist. Cardiac ventricular cells were cultured from chick embryos 14 days in ovo, and the myocyte contractile amplitude was determined as described under “Experimental Procedures.” Cells were exposed to a perfusion medium (4 mm HEPES, pH 7.4, 137 mm NaCl, 3.6 mm KCl, 0.5 mm MgCl2, 1.1 mm CaCl2, 5.5 mm glucose) that contained 2-meSATP (1 μm) in the presence or the absence of the P2 receptor antagonist suramin (50 μm). Measurement of contractile amplitude was carried out on only one cell per coverslip. Both the basal contractile amplitude and the amplitude measured during P2 agonist and antagonist exposure were determined. The representative tracing of eight cells from four cultures is shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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 P2X4receptor in mediating the 2-meSATP-stimulated increase in myocyte contractility, antisense oligonucleotide directed at the 5′-untranslated region contiguous with the translation start site of the cP2X4R was prepared. The antisense oligonucleotide against the cP2X4R effectively blocked the expression of the exogenous cP2X4R when both the oligonucleotide and the cP2X4R cDNA were transduced into the myocyte (Fig.3a). The anti-cP2X4R antibody was unable to detect the native cP2X4R due to a low level expression of the native P2X4R or a low affinity of the antibody for the receptor or both. Therefore, antisense treatment of the myocytes expressing only native P2X4R 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 cP2X4R suggested that the same treatment should also have reduced or eliminated the native P2X4 receptor. Use of the cP2X4R-overexpressing myocyte was to demonstrate that antisense treatment can effectively reduce the level of the receptor. If the native cP2X4R is involved in mediating the 2-meSATP-stimulated 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 agonist-stimulated45Ca influx (−17 ± −7.8%, n = 3, ± S.E.) (Fig. 3, b and c). Similarly, antisense oligonucleotide treatment also blocked the 2-meSATP-stimulated increase in contractile amplitude (3.1 ± 1%, n = 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 ± 13.8 and 61 ± 20% (n = 3, ± S.E.) in the level of45Ca influx in response to 2-meSATP, respectively. Sense oligonucleotide-treated myocytes showed a preserved response to 2-meSATP with a 33 ± 3.3% (n = 10 cells from four cultures) increase in contractile amplitude (Fig. 3, band d). Overall, these data provide more definitive evidence that the P2X4 receptor serves a physiological role, that of mediating an increase in the myocyte contractility and calcium influx in response to ATP. Having established an important physiological role for the P2X4R, its further biochemical characterization was carried out. The cP2X4R detected on the Western blot represented the exogenous recombinant chick P2X4R that was overexpressed following transfection of its cDNA. The anti-cP2X4R antibody detected two major bands, 58- and 44–45-kDa proteins, in an immunoblot of membranes from cP2X4R-overexpressing myocytes (Fig. 3A). The detected molecular mass of the P2X4 receptor in immunoblotting was 58 kDa, consistent with that described by others for the rat P2X4 receptor (20Rubio M.E. Soto F. J. Neurosci. 2001; 21: 641-653Crossref PubMed Google Scholar,21Lê K-T. Villeneuve P. Ramjaun A.R. McPherson P.S. Beaudet A. Séguéla P. Neuroscience. 1998; 83: 177-190Crossref PubMed Scopus (141) Google Scholar). Because the calculated molecular mass of P2X4 receptor is 44–45 kDa, the greater detected molecular mass of 58 kDa may be due to a post-translational modification such as glycosylation, given the presence of seven potential glycosylation sites in the extracellular loop of the P2X4 receptor (16Rettinger J. Aschrafi A. Schmalzing G. J. Biol. Chem. 2000; 275: 33542-33547Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). To test this possibility, the P2X4R-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 P2X4R were treated with tunicamycin (data not shown). These data supported the notion that the P2X4 receptor is glycosylated in cardiac myocytes, similar to the glycosylation of P2X1 and P2X2 receptors (15Torres G.E. Egan T.M. Voigt M.M. Biochemistry. 1998; 37: 14845-14851Crossref PubMed Scopus (58) Google Scholar, 16Rettinger J. Aschrafi A. Schmalzing G. J. Biol. Chem. 2000; 275: 33542-33547Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). At later time points following the transfection with exogenous cP2X4R cDNA, only the 58-kDa receptor protein was present (Fig. 4b), consistent with a more complete glycosylation of the exogenous cP2X4R. Only the 58-kDa P2X4 receptor was detectable in cardiac myocytes isolated from adult wild type mouse and from P2X4receptor transgenic mice, which showed an increased receptor-mediated contractility (11Hu B. Mei Q. Smith E. Barry W.H. Liang B.T. FASEB J. 2001; 10: 2739-2741Google Scholar). These data suggested that the 58-kDa receptor protein, probably representing the glycosylated P2X4receptor, is the final and functional form of the receptor. At the intermediate time point (i.e. 36–48 h following transfection with the cP2X4R 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 P2X4 receptors. To characterize the role of N-linked glycosylation in modulating the biochemical property of the P2X4 receptor, studies were carried out to test the hypothesis that glycosylation is needed for cell surface expression of the P2X4 receptor and for maintaining the detergent solubility of the receptor. Glycosylation 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 P2X4 receptor, cP2X4R-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-cP2X4R antibody showed that tunicamycin could completely block the cell surface expression of P2X4receptor (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 P2X4 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 0.5m 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 P2X4 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 P2X4 receptor and indicated the importance of N-linked glycosylation in maintaining its biochemical property and cell surface expression. We are grateful to Dr. Anja Ruppelt for experimental help and to Dr. Walter Stühmer for generous support.