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-Methylene ATP-Sensitive Phenotype
Glaxo Institute of Applied Pharmacology, Cambridge, United Kingdom (C.A.J., L.A.S., P.P.A.H., I.P.C.); and Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, United Kingdom (C.V., R.J.E.)
Received July 7, 2003; accepted January 14, 2004
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Abstract |
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-methylene ATP. In cells functionally expressing P2X6 receptors, ATP and -methylene ATP evoked slowly desensitizing inward currents (EC50 values, 0.5 and 0.6 µM, respectively) with slow kinetics of current decay on agonist washout. 2',3'-O-(2,4,6-trinitrophenyl ATP) and iso-pyridoxalphosphate-6-azophenyl-2'-5'-disulfonate were effective antagonists (IC50 values, 0.8 and 22 µM, respectively); however, suramin was relatively ineffective. Reverse transcription-polymerase chain reaction analysis confirmed the absence of other P2X receptor subunits. Western analysis of membrane fractions from functional and nonfunctional clones confirmed the presence of P2X6 at the cell membrane but revealed a difference in apparent molecular mass of immunoreactive products (
70 and 60 kDa, respectively). N-glycosidase F treatment of both functional and nonfunctional receptor cell membranes increased the electrophoretic mobilities of immunoreactive products, with both proteins migrating at 55 kDa, demonstrating an increased level of glycosylation of the P2X6 receptor in functional compared with nonfunctional cells. This study demonstrates that nonfunctional rat recombinant P2X6 receptors 1) are expressed on the membrane surface of human embryonic kidney cells and 2) are glycosylated. Expression of the novel functional receptor phenotype is associated with further glycosylation, resulting in an apparently larger molecular mass. These results suggest that P2X6 receptor subunits contribute to -methylene ATP sensitivity.
), the functional phenotypes of several native P2X receptors do not correlate to any recombinant homomeric or heteromeric P2X receptor assembly yet described. For example, responses in the central nervous system are commonly elicited by the ATP analog -methylene-ATP (-meATP) and desensitize relatively slowly (Harms et al., 1992; Chessell et al., 1997; Sansum et al., 1998; Patel et al., 2000). -meATP is a potent full agonist at homomeric and heteromeric receptors that incorporate recombinant P2X1 or P2X3 receptor subunits (Valera et al., 1994). However, it is thought that neither of these subunits is present to a great extent in the central nervous system, and they have been considered unlikely to contribute significantly to -meATP-evoked responses in the central nervous system. In addition, -meATP fails to elicit significant responses from cells expressing recombinant P2X2 (Brake et al., 1994), P2X4 (Bo et al., 1995), P2X5 (Collo et al., 1996), P2X7 (Surprenant et al., 1996), P2X2/6 (King et al., 2000), or P2X4/6 receptors (Lê et al., 1998).
The P2X6 receptor was originally cloned from rat brain and when heterologously expressed in HEK-293 cells was described as being functionally similar to the rat P2X4 receptor (i.e., insensitive to -meATP; Collo et al., 1996). However, other investigators have attempted to replicate these findings but have failed to record ATP-induced responses from recombinant P2X6 receptors expressed in either Xenopus laevis oocytes or HEK-293 cells (Lê et al., 1998; Khakh et al., 1999; Torres et al., 1999). It is worth noting that Collo et al. (1996) reported functional responses from less than 5% of cells transiently transfected with recombinant P2X6, whereas much greater transfection efficiencies were observed when cells were transfected with P2X1-P2X4 receptors. These studies have led to the commonly held view that rat P2X6 receptors do not readily form functional homomeric receptors when expressed in heterologous systems. In some areas of the central nervous system, P2X6 receptors are co-localized with P2X2 and/or P2X4 receptors (Collo et al., 1996; Rubio and Soto, 2001). In these regions, P2X6 receptor subunits may exist as part of heteromeric assemblies of P2X4/6 (Lê et al., 1998), P2X2/6 (King et al., 2000), or potentially P2X2/4/6 and may contribute to the P2X-mediated responses recorded from areas such as the medial habenula (Edwards et al., 1992), locus ceruleus (LC; Harms et al., 1992; Sansum et al., 1998), hippocampus (Wong et al., 2000), and medial vestibular nucleus (Chessell et al., 1997).
In this study, we generated a number of HEK-293 cell lines stably expressing the P2X6 receptor to 1) investigate the mechanisms regulating the expression of functional P2X6 receptors and 2) characterize the pharmacological properties of P2X6 receptors. Our studies demonstrate that the glycosylation state of the receptor is important in determining the production of functional ion channels and suggest that fully glycosylated P2X6 receptor subunits may contribute to -meATP-sensitive P2X receptors.
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Materials and Methods |
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) in addition to the cytomegalovirus promotor. Transfection of HEK-293 Cells. Human embryonic kidney (HEK)-293 cells were transiently transfected with tagged P2X6 receptor cDNA by incubation with 0.5 µg of DNA, 3 µl of LipofectAMINE (Invitrogen), and 200 µl of Dulbecco's modified Eagle's medium (serum free; Invitrogen, Paisley, UK) for 5 h at 37°C. Stable cell lines were created 24 to 48 h after transfection by the addition of G418 (0.6 mg/ml; Invitrogen) or zeocin (0.25 mg/ml; Invitrogen, Groningen, The Netherlands) for cells transfected with pcDNA3.1His or pcDNA4HisMax, respectively. Cells were dilution cloned at least 4 weeks after antibiotic addition and maintained in Dulbecco's modified Eagle's medium/10% fetal bovine serum (Invitrogen, Paisley, UK) in a water-saturated atmosphere of 95% air/5% CO2 at 37°C in 75-cm2 flasks. HEK-293 cells stably expressing P2X2/3, P2X2, or P2X4 receptors were maintained in culture as for stable P2X6 cell lines.
RT-PCR. RNA was prepared using a micro RNA isolation kit (Stratagene, La Jolla, CA) and Reverse-iT 1st Strand synthesis kit (ABgene, Epsom, Surrey, UK). Forward and reverse primers were designed to recognize P2X1-P2X7 receptors. For each reaction, 2 µl of first-strand reaction product was incubated with 400 ng of each primer, 200 µM dNTP, and 5 U of DyNAzyme (Finnzymes Oy, Espoo, Finland) in optimized reaction buffer with a final Mg2+ concentration of 1.5 mM. In each reaction set, PCRs were carried out using both +RT and -RT samples from the reverse transcriptase reactions as well as a no DNA control (in which water was substituted for the first-strand product). Reactions using plasmids containing P2X1-P2X7 as the template (1 µg per reaction) were also carried out to act as positive controls. Reaction products were resolved in a 1% agarose gel.
Western Analysis. Membrane fractions were prepared from cells transfected with P2X6 receptors, resolved on 10% polyacrylamide gels, and electrophoretically transferred onto nitrocellulose (0.22 µm). Blots were blocked overnight at 4°C in Tris-buffered saline supplemented with 0.1% Tween 20 and 5% dried milk. Protein was detected using the antiXpress antibody (1 h, 22°C) and anti-mouse-horseradish peroxidase-conjugated secondary antibody (1 h, 22°C) and visualized using enhanced chemiluminescence detection according to the manufacturer's instructions (Amersham Biosciences, Piscataway, NJ).
Electrophysiology. Whole-cell patch-clamp recordings were made from single cells or small rafts of 3 to 10 cells and voltage clamped at -70 or -90 mV. Cells were continuously perfused with an extracellular solution containing 145 mM NaCl, 2 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, and 10 mM D-glucose (pH 7.3; osmolarity, 300 mOsM). Patch electrodes with resistances of 3 to 5 M
were fire polished and back-filled with internal solution containing either 145 mM cesium aspartate, 11 mM EGTA, 5 mM HEPES, 2 mM NaCl (pH 7.3; osmolarity, 290 mOsM) or 140 mM potassium gluconate, 5 mM NaCl, 10 mM HEPES, or 10 mM EGTA (pH 7.3 adjusted by KOH). Currents were recorded using an Axopatch 200B amplifier (Axon Instruments Inc., Union City, CA) and filtered with a corner frequency of 1 to 5 kHz (8-pole Bessel filter), digitized at 2 to 10 kHz using a Digidata 1200A (Axon Instruments Inc.) interface, and stored on a computer. For the construction of concentration-effect curves, 1- to 2-s applications of agonist (ATP or -meATP; Sigma Chemical, Poole, Dorset, UK) were separated by wash periods of 5 (P2X4) or 2 min (P2X2). P2X6 receptor-mediated responses had prolonged time courses, and the rate of closure of the channels after washout of agonists was considerably prolonged (see Results); to get complete return to baseline current and record reproducible responses, an interval of 10 to 15 min was required between agonist applications. For concentration-response data, responses (mean ± S.E.M) are expressed as a percentage of the maximum response to 100 µM ATP. For antagonist experiments, the cells were presuperfused with antagonist solution for 10 min before the concomitant application of ATP (3 µM; an EC90 concentration) and the antagonist. In all experiments, drugs were applied using a computer controlled fast-flow U-tube application system (Nakazawa and Ohno, 1997). 2',3'-O-(2,4,6-trinitrophenyl ATP) (TNP-ATP) was purchased from Molecular Probes (Eugene, OR), and iso-pyridoxalphosphate-6-azophenyl-2'-5'-disulfonate (iso-PPADS) and suramin were obtained from Sigma Chemical.
N-Glycosidase F Treatment. For deglycosylation of recombinant P2X6 receptor protein expressed in HEK-293 cells, an N-glycosidase F deglycosylation kit (Roche Applied Science, Indianapolis, IN) was employed. Cell membranes were prepared as described previously (Castro et al., 1996), and protein levels per sample were determined using the copper reduction/bicinchoninic acid protein assay (Pierce Chemical, Rockford, IL) as per the manufacturer's instructions. One hundred micrograms of protein was used for each deglycosylation reaction. Samples were incubated at 95°C for 3 min with reduced denaturing buffer (20 mM phosphate-buffered saline, 1% sodium dodecyl sulfate, and 1% 2-mercaptoethanol; pH 8.6) to denature the proteins. N-glycosidase F (2.4 U per sample) and reaction buffer (20 mM phosphate-buffered saline, 10 mM EDTA, and 0.5% n-octylglycopyranoside; pH 7.2) were added, and the tubes were incubated at 37°C for 1 h. Reactions were terminated by the addition of Laemmli sample buffer, and samples were analyzed on 10% polyacrylamide gels (see above).
Data Analysis. Where appropriate, concentration-effect curves were fitted using a three-parameter logistic equation (GraphPad Prism; GraphPad Software Inc., San Diego, CA). EC50 and IC50 values are expressed as geometric mean ± 95% confidence intervals, whereas individual data are expressed as mean ± S.E.M. Statistical comparisons were made using the Student's paired or unpaired t test where appropriate, and the null hypothesis was rejected when P < 0.05.
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Results |
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), functional responses were recorded from less than 5% of cells tested. Two cell lines were selected for further investigation, one expressing functional P2X6 and one expressing nonfunctional P2X6 receptor protein, both transfected with the P2X6/pcDNA4HisMax construct.
Functional and Nonfunctional P2X6 Receptors Are Differentially Glycosylated. Western analysis of membrane preparations from the functional and nonfunctional P2X6 stable cell lines confirmed the presence of recombinant P2X6 receptors in membrane fractions of both lines. However, immunoreactive products from functional and nonfunctional P2X6 cells had different apparent molecular masses (70 and 60 kDa, respectively; Fig. 1A). Figure 1A also shows the immunoreactive products identified from membrane fractions of cells transiently transfected with recombinant P2X6 receptors and representing a mixed population of cells. In this case, three bands are clearly visible at approximately 70, 60, and 50 kDa.
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Treatment of P2X6 cell membranes with N-glycosidase led to a decrease in the apparent molecular mass of the 70-kDa product to 55 kDa. The band at 62 kDa observed in the nonfunctional P2X6 membrane preparation was also shifted in size to 55 kDa after deglycosylation, and an additional product of 50 kDa was apparent (Fig. 1B). This would suggest that functional P2X6 and nonfunctional P2X6-expressing cells differentially glycosylate the P2X6 receptor.
P2X6 Receptors Have a Novel Phenotype. Whole-cell patch clamp was used to functionally characterize the P2X6-mediated currents. ATP induced rapid inward currents in all of the cells tested. The current-voltage relationship was typical of a nonselective cation channel, with a reversal potential of approximately zero (-4.8 mV with cesium aspartate internal solution) and showed a slightly inwardly rectifying profile (Fig. 2D). Little desensitization was observed with agonist applications of up to 2 s (87 ± 5% response remaining after 2-s application of 30 µM ATP, n = 10). Currents recorded in P2X6 cells were found to return slowly to baseline levels: the time for the current to ATP (3 µM) to decay by 50% at the end of agonist application was 8.7 ± 0.6 s (n = 5), and return to baseline current usually took >1 min. This rate of decay of the current is significantly prolonged compared with application of ATP (3 µM) to recombinant P2X2/3 receptors (time to 50% decay at the end of the pulse, 0.8 ± 0.2 s; n = 5; P < 0.05; Fig. 2C). Mean maximum current amplitudes were smaller from cells expressing P2X6 receptors (420 ± 31 pA, n = 50) compared with P2X2/3 receptors (4101 ± 790 pA, n = 5) but were larger than those from cells expressing P2X4 receptors (258 ± 40, n = 29).
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Concentration-effect curves were determined for two P2 receptor agonists, ATP and -meATP, to allow comparison between the P2X6 receptor-mediated currents with those recorded from native tissue and also with previously characterized P2X receptors. ATP was found to be significantly more potent at P2X6 [EC50 = 0.5 (0.4-0.6) µM, n = 6] than at P2X4 [EC50 = 5.5 (2.6-11.6) µM, n = 7] or P2X2 receptors]EC50 = 33 (30-36) µM, n = 6; Fig. 2]. -meATP, the ATP analog that has been widely used in the study of native responses, was found to act as a full agonist with respect to ATP at cells expressing P2X6 receptors (Fig. 3). -meATP had equivalent potency with ATP at P2X6 receptors [EC50 = 0.6 (0.4-1.0) µM, n = 3], which is in stark contrast to its lack of effect at cells expressing rat P2X4 receptors. It is also interesting to note that the time course of the decay of P2X6 currents on removal of agonist was faster for -meATP compared with an equivalent concentration (3 µM) of ATP (5.1 ± 0.6 s versus 8.7 ± 0.6 s, respectively; n = 5 for each, P < 0.05; Figs. 2 and 3). The agonists ,-methylene ADP and benzoyl benzoyl ATP were inactive at P2X6 receptors at concentrations up to 100 µM (data not shown).
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We determined the effect of three P2X receptor antagonists on responses to an EC90 concentration of ATP (3 µM; Fig. 4). TNP-ATP and iso-PPADS were effective antagonists of the ATP response with IC50 values of 0.83 (0.16-4.4) and 21.8 (11.3-41.9) µM, respectively. In contrast, the response to ATP was relatively suramin-insensitive with only a 27 ± 4.6% reduction in response (n = 4) to the maximal concentration of suramin tested (100 µM). Similar antagonist effects were seen when -meATP (3 µM) was the agonist (80.9 ± 6% inhibition by 10 µM TNP-ATP, 89.2 ± 0.8% inhibition by 100 µM iso-PPADS, and 30.5 ± 2.4% inhibition by 100 µM suramin, n = 3 for each).
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P2X6-Expressing Cells Do Not Contain Other P2X Subunits. The observed pharmacological differences between responses in P2X6-expressing cells compared with those reported previously (Collo et al., 1996) led to concerns that the responses we describe could be caused by receptors other than homomeric P2X6. It has been suggested that under certain conditions, HEK-293 cells can endogenously express certain P2X receptor subunits (Worthington et al., 1999b). It was thus theoretically possible that heteropolymerization between endogenous P2X receptors with recombinant P2X6 could form a novel heteromeric P2X receptor responsible for P2X6 responses to ATP. With this in mind, RT-PCRs were carried out on P2X6-expressing cells (Fig. 5). Positive controls (pcDNA3 plasmids containing cDNA encoding each of the individual receptor subunits) show the successful completion of each PCR, whereas negative controls demonstrate a lack of genomic contamination of P2X6 cell DNA samples (-RT control) and a lack of external contamination in the reactions (no DNA control). RT-PCRs detected the presence of mRNA encoding P2X6 but failed to detect the presence of mRNA for any of the other known P2X receptors. Immunocytochemical studies using the commercially available antibodies against P2X receptors confirmed the absence of P2X1, P2X2, P2X3, P2X4, and P2X7 (data not shown).
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Discussion |
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) and for P2X6 yields a slowly desensitizing, -meATP-sensitive phenotype.
Few data exist on the expression and properties of recombinant homomeric P2X6 receptors, because functional responses to ATP have proved elusive after transient expression of the protein in heterologous systems. It has been suggested that P2X6 is poorly expressed in non-native systems but here, we show that although transient transfection efficiencies were variable and lower than expected, recombinant, albeit frequently nonfunctional P2X6 receptors can be expressed in stable cell lines. In line with the original study (Collo et al., 1996), functional responses were recorded from less than 5% of cells expressing recombinant P2X6 receptor protein. Previous studies have not investigated the membrane localization of P2X6 receptors in recombinant systems, partly because of the absence of a reliable antibody against P2X6. As demonstrated in this study (Fig. 1), the dominant immunoreactive product detected using Western analysis of transiently transfected cells had an apparent molecular mass of 50 kDa, the expected size of a nonglycosylated P2X6 receptor subunit, based on primary sequence. In this study, we have provided evidence that at least partial glycosylation of the receptor is required for cell membrane expression and that expression of a functional phenotype is associated with further glycosylation, explaining the absence of functional responses in the majority of cells transiently expressing recombinant P2X6 receptor protein.
P2X6 receptor protein has been detected as an 49-kDa band from whole rat brain homogenates (5 days postnatal; Rubio and Soto, 2001) and as an 70-kDa product from adult rat midbrain homogenate (Worthington et al., 1999a), atrium, bladder, kidney (Worthington et al., 1999b), and thymocytes (Glass et al., 2000). It has also been reported at 60 kDa from human superior cervical ganglia, HEK-293 cells, rat ventricle, bladder, and kidney (Worthington et al., 1999b). Yunaev et al. (2000) also report P2X6 immunoreactive products running at 50, 55, 60, and 70 kDa from the rat urinary bladder, so it seems possible that the recombinant P2X6 species detected in stable P2X6 receptor cell lines and transiently transfected cells may have physiological correlates in some tissues. To resolve this issue, a detailed study of P2X6 receptor expression and glycosylation status in different brain regions and in animals of different ages is required. Age-related differences in P2X-mediated currents have been reported from the LC (Wirkner et al., 1998) in which 2-methylthioATP-induced currents increase with age, reaching maturity at 18 days postnatal. It is therefore possible that the 49-kDa P2X6 receptor protein detected from rat brain (Rubio and Soto, 2001) represents an immature form of the receptor and that increased levels of glycosylation and increased function occur with age.
The finding that P2X6 receptors exist in a number of different glycosylation states is consistent with previous work carried out on recombinant P2X1 (Nicke et al., 1998; Rettinger et al., 2000), recombinant P2X2 (Newbolt et al., 1998; Torres et al., 1998a,b), and cardiac P2X4 receptors (Hu et al., 2002). The P2X1 subunit possesses five putative N-glycosylation sites (Asn-X-Ser, where X is any amino acid except proline). The elimination of three of the four usable sites leads to impaired protein expression, whereas elimination of all four abolished expression of the receptor (Newbolt et al., 1998). The P2X6 sequence has three putative sites for N-linked glycosylation at amino acids 157, 187, and 202. In the present study, nonfunctional P2X6 receptor protein was found to be glycosylated and at the cell membrane. This presence of N-linked glycans on nonfunctional receptors is thus in keeping with previous reports, which suggest that a minimum amount of glycosylation is absolutely required for P2X receptor expression at the cell surface; the molecular correlates of these sites remain to be determined.
One question remains: why do functional recombinant P2X6 receptors only form in a small proportion of cells? It is clear that the glycosylation state plays a key role in receptor function, but the factors that regulate the degree of glycosylation remain to be determined. It is interesting that Torres et al. (1999) reported that coimmunoprecipitation of P2X6 receptor subunits was below the limit of detection but P2X6 receptors could be coimmunoprecipitated with P2X1, P2X2, P2X4, and P2X5 receptor subunits, suggesting that these other subunits could assist in the correct assembly of the receptor. The low rate of functional P2X6 receptors could theoretically have been caused by heteropolymerization with endogenously expressed P2X4 receptors that have been reported in a small number of HEK-293 cells (Worthington et al., 1999b). However, in the present study, non-P2X6 receptor subunits were below the limit of detection by RT-PCR or immunohistochemically. It thus seems unlikely that these non-P2X6 receptor subunits contribute to the observed phenotype of P2X6 receptors. However, it is possible that an additional auxiliary subunit is required for the functional expression of P2X6 receptors.
Pharmacological characterization of recombinant P2X6 receptors revealed a novel phenotype. This was significantly different from that reported in the only other study where recombinant P2X6 receptors have been described previously (Collo et al., 1996). In particular, the responses differ in terms of time course and sensitivity to -meATP. Responses reported by Collo et al. (1996) are very similar to those of rat recombinant P2X4 receptors. P2X4 receptor RNA and protein can be expressed in native HEK-293 cells; however, no one has reported P2X-like responses from nontransfected HEK-293 cells (North, 2002). This raises the possibility that in the study of Collo et al. (1996), endogenous P2X4 receptor subunits may have combined with P2X6 receptors to form functional channels. Alternatively, differential glycosylation, post-translational processing, or the action of auxiliary subunits may account for the differences in properties between the work of Collo et al. (1996) and the present study.
In the present study, ATP (100 µM) induced inward currents and was found to be significantly more potent at rat P2X6 receptors than at rat P2X4 or P2X2 receptors. P2X6 receptor responses were relatively nondesensitizing during agonist application; however, the time taken for the response to return to baseline on agonist washout was considerably prolonged compared with most other P2X receptors and was similar to that described for P2X7 receptors (Rassendren et al., 1997). In addition, the rate of response decline was also dependent on the agonist activating the receptor. The mechanism for the slow recovery on agonist washout and the agonist-specific variations remain to be determined but could result from a long mean open time of the P2X6 receptor channel, very slow unbinding rate of the agonist, or, as for the P2X7 receptor, the formation of a dilating pore. P2X6 receptors were also found to be highly sensitive to the ATP analog, -meATP, which acted as a full agonist with respect to ATP. To date, only homomeric P2X1 (Valera et al., 1994), P2X3 (Chen et al., 1995), and heteromeric P2X2/3 (Lewis et al., 1995) and P2X1/5 (Lê et al., 1999) have been described as sensitive to this agonist. P2X4/6 receptors coexpressed in X. laevis oocytes were reported to show an increased sensitivity to -meATP compared with homomeric P2X4 receptors (Lê et al., 1998), but the increase was small and in our own studies, found to be nonsignificant (Jones et al., 2001). This finding is therefore potentially very important, and the expression of P2X6 receptor subunits could explain the incidence of -meATP-induced responses from brain areas in which P2X1 and P2X3 receptors are lacking. However, two pieces of evidence suggest that it is unlikely that the majority of -meATP-sensitive brain P2X receptors correspond to homomeric P2X6 receptors. First, in the present study, P2X6 receptors were relatively insensitive to the antagonist suramin, which has proved effective at many neuronal P2X receptors (e.g., Edwards et al., 1992; Chessell et al., 1997; Sansum et al., 1998); and second, P2X6 receptors were insensitive to -methylene ADP, the ATP analog shown to elicit inward currents in the LC (Sansum et al., 1998; Patel et al., 2000) and medial vestibular nucleus (Chessell et al., 1997). Therefore, although P2X6 receptors may contribute -meATP sensitivity to heteromeric channels in the central nervous system, the subunit stoichiometry of these remains to be elucidated.
Overall, this study has described the first investigation of functional homomeric P2X6 receptors stably expressed in HEK-293 cells and provides evidence that nonfunctional P2X6 receptors are expressed at the cell surface and are glycosylated. We suggest that although the presence of a minimum number of N-glycans is required for receptor expression, further glycosylation is required to confer subunit function. Finally, functional homomeric P2X6 receptors were found to be slowly desensitizing and sensitive to the ATP analog, -meATP, and thus show a novel operational phenotype. We propose that slowly desensitizing -meATP-mediated responses in tissue lacking P2X1 and P2X3 receptors could be accounted for by the contribution of fully glycosylated P2X6 receptor subunits to heteromeric channels.
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Footnotes |
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ABBREVIATIONS: -meATP, -methylene ATP; HEK, human embryonic kidney; PPADS, pyridoxalphosphate-6-azophenyl-2'-5'-disulfonate; LC, locus ceruleus; TNP-ATP, 2',3'-O-(2,4,6-trinitrophenyl ATP); RT, reverse transcription; PCR, polymerase chain reaction.
1 Current address: ITIS UMR 6150 CNRS, Bd Pierre Dramard, 13916 Marseille cedex 20, France. 
2 Current address: Department of Neurobiology, The Babraham Institute, Cambridge, CB2 4AT, UK.
3 Current address: Advanced Medicine, Inc., 901 Gateway Boulevard, South San Francisco, CA 94080.
4 Current address: Neurology CEDD, GlaxoSmithKline, New Frontiers Science Park-North, Coldharbour Road, The Pinnacles, Harlow, Essex, CM19 5AW, UK.
Address correspondence to: Iain Chessell, Neurology CEDD, GlaxoSmithKline, New Frontiers Science Park-North, Coldharbour Road, The Pinnacles, Harlow, Essex CM19 5AW, UK. E-mail: iain.p.chessell{at}gsk.com
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Brake AJ, Wagenbach MJ, and Julius D (1994) New structural motif for ligand-gated ion channels defined by an ionotropic ATP receptor. Nature (Lond) 371: 519-523.[CrossRef][Medline]
Castro SW, Buell G, Feniuk W, and Humphrey PP (1996) Differences in the operational characteristics of the human recombinant somatostatin receptor types, sst1 and sst2 in mouse fibroblast (Ltk-) cells. Br J Pharmacol 117: 639-646.[Medline]
Chen CC, Akopian AN, Sivilotti L, and Colquhoun D (1995) A P2X purinoceptor expressed by a subset of sensory neurons. Nature (Lond) 377: 428-431.[CrossRef][Medline]
Chessell IP, Michel AD, and Humphrey PP (1997) Functional evidence for multiple purinoceptor subtypes in the rat medial vestibular nucleus. Neuroscience 77: 783-791.[CrossRef][Medline]
Collo G, North RA, Kawashima E, Merlo-Pich E, Neidhart S, and Surprenant A (1996) Cloning of P2X5 and P2X6 receptors and the distribution and properties of an extended family of ATP-gated ion channels. J Neurosci 16: 2495-2507.
Edwards FA, Gibb AJ, and Colquhoun D (1992) ATP receptor-mediated synaptic currents in the central nervous system. Nature (Lond) 359: 144-147.[CrossRef][Medline]
Glass R, Townsend-Nicholson A, and Burnstock G (2000) P2 receptors in the thymus: expression of P2X and P2Y receptors in adult rats, an immunohistochemical and in situ hybridisation study. Cell Tissue Res 300: 295-306.[CrossRef][Medline]
Harms L, Finta EP, Tschöpl M, and Illes P (1992) Depolarization of rat locus coeruleus neurons by adenosine 5'-triphosphate. Neuroscience 48: 941-952.[CrossRef][Medline]
Hu B, Senkler C, Yang A, Soto F, and Liang BT (2002) P2X4 receptor is a glycosylated cardiac receptor mediating a positive inotropic response to ATP. J Biol Chem 277: 15752-15757.
Jones CA, Simon J, Humphrey PPA, and Chessell IP (2001) Co-expression and functional characterisation of rat P2X4 and P2X6 subunits in mammalian cells Br J Pharmacol 134: 46P.[CrossRef]
Khakh BS, Proctor WR, Dunwiddie TV, Labarca C, and Lester HA (1999) Allosteric control of gating and kinetics at P2X4 receptor channels. J Neurosci 19: 7289-7299.
King BF, Townsend-Nicholson A, Wildman SS, Thomas T, Spyer KM, and Burnstock G (2000) Coexpression of rat P2X2 and P2X6 subunits in Xenopus oocytes. J Neurosci 20: 4871-4877.
Lê KT, Babinski K, and Seguela P (1998) Central P2X4 and P2X6 channel subunits coassemble into a novel heteromeric ATP receptor. J Neurosci 18: 7152-7159.
Lê KT, Boue-Grabot E, Archambault V, and Seguela P (1999) Functional and biochemical evidence for heteromeric ATP-gated channels composed of P2X1 and P2X5 subunits. J Biol Chem 274: 15415-15419.
Lewis C, Neidhart S, Holy C, North RA, Buell G, and Surprenant A (1995) Coexpression of P2X2 and P2X3 receptor subunits can account for ATP-gated currents in sensory neurons. Nature (Lond) 377: 432-435.[CrossRef][Medline]
Nakazawa K and Ohno Y (1997) Effects of neuroamines and divalent cations on cloned and mutated ATP-gated channels. Eur J Pharmacol 325: 101-108.[CrossRef][Medline]
Newbolt A, Stoop R, Virginio C, Surprenant A, and North RA (1998) Membrane topology of an ATP-gated ion channel (P2X receptor). J Biol Chem 273: 15177-15182.
Nicke A, Baumert HG, Rettinger J, Eichele A, and Lambrecht (1998) P2X1 and P2X3 receptors form stable trimers: a novel structural motif of ligand-gated ion channels. EMBO (Eur Mol Biol Organ) J 17: 3016-3028.[CrossRef][Medline]
North RA (2002) Molecular physiology of P2X receptors. Physiol Rev 82: 1013-1067.
Patel MK, Khakh BS, and Henderson G (2000) Properties of native P2X receptors in rat trigeminal mesencephalic nucleus neurones: lack of correlation with known, heterologously expressed P2X receptors. Neuropharmacology 40: 96-105.
Rassendren F, Buell GN, Virginio C, Collo G, North RA, and Surprenant A (1997) The permeabilizing ATP receptor, P2X7. Cloning and expression of a human cDNA. J Biol Chem 272: 5482-5486.
Rettinger J, Aschrafi A, and Schmalzing G (2000) Roles of individual N-glycans for ATP potency and expression of the rat P2X1 receptor. J Biol Chem 275: 33542-33547.
Rubio ME and Soto F (2001) Distinct localization of P2X receptors at excitatory postsynaptic specializations. J Neurosci 21: 641-653.
Sansum AJ, Chessell IP, Hicks GA, Trezise DJ, and Humphrey PP (1998) Evidence that P2X purinoceptors mediate the excitatory effects of methylene-ADP in rat locus coeruleus neurones. Neuropharmacology 37: 875-885.[CrossRef][Medline]
Surprenant A, Rassendren F, Kawashima E, North RA, and Buell G (1996) The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7). Science (Wash DC) 272: 735-738.[Abstract]
Stein I, Itin A, Einat P, Skaliter R, Grossman Z, and Keshet E (1998) Translation of vascular endothelial growth factor by mRNA by internal ribosome entry: implications for translation under hypoxia. Mol Cell Biol 18: 3112-3119.
Torres GE, Egan TM, and Voigt MM (1998a) N-linked glycosylation is essential for the functional expression for the recombinant P2X2 receptor. Biochemistry 37: 14845-14851.[CrossRef][Medline]
Torres GE, Egan TM, and Voigt MM (1998b) Topological analysis of the ATP-gated ionotropic P2X2 receptor subunit. FEBS Lett 425: 19-23.[CrossRef][Medline]
Torres GE, Egan TM, and Voigt MM (1999) Hetero-oligomeric assembly of P2X receptor subunits. Specificities exist with regard to possible partners. J Biol Chem 274: 6653-6659.
Valera S, Hussy N, Evans RJ, Adami N, North RA, Surprenant A, and Buell G (1994) A new class of ligand-gated ion channel defined by P2X receptor for extracellular ATP. Nature (Lond) 371: 516-519.[CrossRef][Medline]
Wirkner K, Franke H, Inoue K, and Illes P (1998) Differential age-dependent expression of 2 adrenoceptor- and P2 purinoceptor-functions in rat locus coeruleus neurons. Naunyn-Schmiedeberg's Arch Pharmacol 357: 186-189.[CrossRef][Medline]
Wong AYC, Burnstock G, and Gibb AJ (2000) Single channel properties of P2X ATP receptors in outside-out patches from rat hippocampal granule cells. J Physiol (Lond) 527: 529-547.
Worthington RA, Arumugam TV, Hansen MA, Balcar VJ, and Barden JA (1999a) Identification and localisation of ATP P2X receptors in rat midbrain. Electrophoresis 20: 2077-2080.[CrossRef][Medline]
Worthington RA, Dutton JL, Poronnik P, Bennett MR, and Barden JA (1999b) Localisation of P2X receptors in human salivary gland epithelial cells and human embryonic kidney cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/Western blotting and immunofluorescence. Electrophoresis 20: 2065-2070.[CrossRef][Medline]
Yunaev MA, Barden JA, and Bennett MR (2000) Changes in the distribution of different subtypes of P2X receptor clusters on smooth muscle cells in relation to nerve varicosities in the pregnant rat urinary bladder. J Neurocytol 29: 99-108.[CrossRef][Medline]
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) compared with rat P2X4 (
)and rat P2X2 (
) receptors (A). Serially increasing concentrations of ATP were applied for 1 s at 15- (P2X6, n = 6), 5- (P2X4 n = 7), or 2-min (P2X2, n = 6) intervals. Data are mean ± S.E.M. from n experiments. A representative trace from cells expressing rat P2X6 receptors is given (B). P2X6 receptors show a very slow recovery to baseline current on removal of agonist (C). A representative trace shows the off rate after ATP (3 µM) application, normalized to the peak response for P2X6- and P2X2/3-expressing cell lines (C) The voltage-current relationship for P2X6 receptors is also shown (D). Voltage ramps from -90 to +60 mV were carried out on single cells using the whole-cell patch-clamp technique. Data are mean ± S.E.M. from four experiments.
