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. 1998 Jul 1;510(Pt 1):27–35. doi: 10.1111/j.1469-7793.1998.027bz.x

Calcium permeability and block at homomeric and heteromeric P2X2 and P2X3 receptors, and P2X receptors in rat nodose neurones

Caterina Virginio 1, R A North 1, Annmarie Surprenant 1
PMCID: PMC2231016  PMID: 9625864

Abstract

  1. Whole-cell recordings were made from HEK 293 (human embryonic kidney) cells stably transfected with cDNAs encoding P2X2, P2X3 or both receptors (P2X2/3) and from cultured rat nodose neurones. Nodose neurones all showed immunoreactivity for both P2X2 and P2X3, but not P2X1, receptors.

  2. Reversal potentials were measured in extracellular sodium, N-methyl-D-glucamine (NMDG) and NMDG containing 5 mM Ca2+; the values were used to compute relative permeabilities (PNMDG/PNa and PCa/PNa). PNMDG/PNa was not different for P2X2, P2X2/3 and nodose neurones (0.03) but was significantly higher (0.07) for P2X3 receptors. PCa/PNa was not different among P2X3, P2X2/3 and nodose neurones (1.2-1.5) but was significantly higher (2.5) for P2X2 receptors.

  3. External Ca2+ inhibited purinoceptor currents with half-maximal concentrations of 5 mM at the P2X2 receptor, 89 mM at the P2X3 receptor and 15 mM at both the P2X2/3 heteromeric receptor and nodose neurones. In each case, the inhibition was voltage independent and was overcome by increasing concentrations of agonist.

  4. These results may indicate that Ca2+ permeability of the heteromeric (P2X2/3) channel is dominated by that of the P2X3 subunit, while Ca2+ block of the receptor involves both P2X2 and P2X3 subunits. The correspondence in properties between P2X2/3 receptors and nodose ganglion neurones further supports the conclusion that the native α,β-methylene ATP-sensitive receptor is a P2X2/3 heteromultimer.

ATP-gated ion channels, the P2X receptors, are present in the cell bodies as well as both peripheral and central terminals of most sensory neurones (Vulchanova et al. 1996, 1997; Collo et al. 1996), where they may play a role in proprioception, nociception and/or transmission of primary afferent information (Evans & Surprenant, 1996; Burnstock, 1996). Of the seven P2X receptors (see North & Barnard, 1997), six are present in sensory ganglia (P2X1-P2X6), generally with distinct patterns (Valera et al. 1994; Brake, Wagenbach & Julius, 1994; Chen, Akopian, Sivilotti, Colquhoun, Burnstock & Wood, 1995; Lewis, Neidhart, Holy, North, Buell & Surprenant, 1995; Buell, Lewis, Collo, North & Surprenant, 1996; Collo et al. 1996). For example, P2X5 mRNA is observed in a population of large-diameter dorsal root ganglia (DRG) neurones while P2X1, P2X2, P2X4 and P2X6 mRNAs can be detected in both large- and small-diameter neurones, and P2X3 mRNA appears to be restricted to a subpopulation of small-diameter neurones (Chen et al. 1995; Collo et al. 1996; Vulchanova et al. 1996, 1997). Immunohistochemical studies have revealed a particularly close relationship between P2X2 and P2X3 receptor protein in a subpopulation of small-diameter, unmyelinated neurones in sensory dorsal root, trigeminal and nodose ganglia (Vulchanova et al. 1997). Moreover, P2X3 immunoreactivity has been demonstrated in both the soma and sensory endings of functionally identified nociceptor neurones in trigeminal ganglia (Cook, Vulchanova, Hargreaves, Elde & McCleskey, 1997).

Both P2X2 and P2X3 cDNAs result in the expression of robust, homomeric cation-selective ionotropic channels when expressed in oocytes or mammalian cell lines (Brake et al. 1994; Lewis et al. 1995; Chen et al. 1995). The homomeric P2X2 channel is most readily characterized by its insensitivity to activation by the ATP analogue, α,β-methylene ATP (α,β-meATP), and its relative lack of desensitization during ATP applications of up to 10 s; the homomeric P2X3 channel shows the converse characteristics (i.e. α,β-meATP is a potent agonist and the current rapidly desensitizes within 0.5-2 s) (Brake et al. 1994; Lewis et al. 1995; Chen et al. 1995; Evans & Surprenant, 1996; Garcia-Guzman, Stuhmer & Soto, 1997). In addition, these two subunits readily heteropolymerize to form receptors exhibiting an α,β-meATP-sensitive, non-desensitizing phenotype (Lewis et al. 1995; Radford, Virginio, Surprenant, North & Kawashima, 1997). These two properties (α,β-meATP sensitivity and desensitization) allow the responses of sensory neurones to be divided into two classes: they resemble either the homomeric P2X3 phenotype or the heteromeric P2X2/3 phenotype (Krishtal, Marchenko, Obukhov & Volkova, 1988; Bean, 1992; Khakh, Humphrey & Surprenant, 1995; Lewis et al. 1995; Robertson, Rae, Rown & Kennedy, 1996; Evans & Surprenant, 1996; Cook et al. 1997). A comparison of other physiological properties among homomeric P2X2, homomeric P2X3, heteromeric P2X2/3 receptors and native P2X receptors in sensory neurones will be valuable in efforts to assign a molecular identity to the sensory neurone receptors.

P2X receptors in some sensory neurones are permeable to calcium (Bean, Williams & Ceelen, 1990). In other cells, calcium can permeate the channels (e.g. smooth muscle: Benham & Tsien, 1987) or block them (e.g. phaeochromocytoma (PC12) cells: Nakazawa & Hess, 1993). We have shown previously that P2X1 and P2X2 receptors, heterologously expressed, differ in both calcium permeability and block (Evans et al. 1996). The main purpose of the present work was to examine calcium permeability and block at heterologously expressed P2X2, P2X3 and P2X2/3 receptors, and to compare these with the properties of the α,β-meATP-sensitive P2X receptors expressed in nodose ganglion neurones.

METHODS

Preparation of cells

HEK 293 (human embryonic kidney) cells stably expressing the rat P2X2 receptor have been described previously (Evans et al. 1996). A similar procedure was followed to obtain HEK 293 cells stably expressing the P2X3 receptor. Briefly, complementary DNA (cDNA) encoding the rat P2X3 receptor was subcloned in the pcDNA3 vector (Stratagene) and 5 × 106 HEK 293 cells were electroporated with 30 μg of P2X3 cDNA; 48 h later selection was begun using G418 (800 μg ml−1; geneticin sulphate, Gibco). Positive clones were selected by recording currents evoked by ATP or α,β-meATP; the most responsive clone was thereafter maintained in 300 μg ml−1 G418 and used for all experiments. The HEK 293 cell line stably expressing the heteromeric P2X2/3 receptor using internal ribosome entry site construction has been described in detail previously (Kawashima, Estoppey, Virginio, Rees, Surprenant & North, 1998). Dissociation and culture conditions for rat nodose neurones were followed as described in detail previously (Stansfield & Mathie, 1993; Khakh et al. 1995). Young adult (3-4 weeks) rats were anaesthetized under halothane and guillotined; these methods have been approved by the Office Veterinaire Cantonal of Geneva. All recordings were obtained from neurones maintained for 4-12 days in culture.

Electrophysiological recordings

Whole-cell patch clamp recordings were carried out using an Axopatch 200A; pCLAMP 6 and AxoGraph 3 (Axon Instruments) software were used for data acquisition and analysis. Patch pipettes (4-7 MΩ) contained (mM): NaCl, 154; EGTA, 10; and Hepes, 10. Gigaohm seals and whole-cell configuration were established using standard external solution containing (mM): NaCl, 147; KCl, 2; MgCl2, 1; CaCl2, 2; Hepes, 10; and glucose, 12; the external solution also contained tetrodotoxin (1–10 μM, Sigma) for experiments on nodose neurones in order to block sodium currents. All experiments were carried out at room temperature (22-24°C). The following protocol was used for reversal potential measurements: Five to ten minutes after establishing whole-cell configuration, the external solution was changed to (mM): NaCl, 154; Hepes, 10; glucose, 12; and reversal potential (Vrev) determined. External solution was then changed to (mM): N-methyl-D-glucosamine (NMDG), 154; Hepes, 10; glucose, 12; and Vrev determined. Next, 5 mM CaCl2 was added to the NMDG solution and Vrev again determined; an equal number of experiments were carried out using the reverse order of solutions (i.e. NMDG-Ca2+ followed by NMDG only). Osmolarity and pH of all solutions were maintained at 300-315 mosmol l−1 and 7.3, respectively. In each solution the membrane was held at four to eight different potentials within ±15 mV of the reversal potential and ATP or α,β-meATP applied (1-2 s duration) by the U-tube method (Fenwick, Marty & Neher, 1982), at intervals of 30 s for experiments on heterologously expressed P2X2 and P2X2/3 receptors and nodose neurones, or at 2 min intervals in the case of expressed P2X3 receptors because of the strong run-down of this current upon repeated applications (Chen et al. 1995; Lewis et al. 1995). We have previously carried out experiments to determine NMDG and/or Ca2+ permeability ratios for the homomeric P2X2 and homomeric P2X3 receptor (Lewis et al. 1995; Evans et al. 1996); however, the ionic compositions of the solutions used were different; therefore we now repeated these measurements using solutions described herein so that we could make direct comparisons for all receptors studied.

PNMDG/PNa was calculated from: exp(ΔVrevF/RT). PCa/PNa was calculated from:

graphic file with name tjp0510-0027-mu1.jpg

Calculations were made using ion activities of 0.75 for sodium and NMDG and 0.3 for calcium (Benham & Tsien, 1987; Otis, Raman & Trussell, 1995; Koh, Geiger, Jonas & Sakmann, 1995). For studies on the inhibition of purinoceptor currents by calcium, standard external solution was used with isosmotic substitution of CaCl2 for NaCl. Sodium salts of ATP and α,β-methylene ATP (α,β-meATP, Sigma) were used in all experiments. Results are plotted as means ± s.e.m. Student's t test or the Mann-Whitney non-parametric test was used for comparisons; P values ≤ 0.01 were considered significant.

Immunohistochemistry

The immunohistochemical double-labelling techniques and antisera used are detailed in previous publications (Vulchanova et al. 1996, 1997). Briefly, cells were fixed with Zamboni's solution (4 % paraformaldehyde and 0.2 % picric acid in 0.1 M phosphate buffer, pH 6.9) for 20 min, rinsed and stored in physiological saline solution. Rabbit anti-P2X2 antibody and guinea-pig anti-P2X3 antiserum were used at 1:1000 or 1:2000 dilution. Secondary antibodies, fluorescein isothiocyanate (FITC)-conjugated donkey anti-guinea-pig immunoglobulin G (IgG) and tetramethyl rhodamine isothiocyanate (TRITC)-conjugated donkey anti-rabbit IgG, were used at 1:100 dilution. Control preparations were treated with primary antibodies preincubated with their cognate peptide (10 μM) (Vulchanova et al. 1996). Pseudo-colour confocal laser microscopy (Zeiss LSM 410 microscope) was used for digital imaging of immunofluorescent cells; images were stored as TIF files and subsequently printed directly on an Agfa Duoproof colour printer.

RESULTS

General observations

In agreement with previous immunohistochemical studies of rat nodose neurones in situ (Vulchanova et al. 1996, 1997), cultured nodose neurones used in the present study were also positive for both P2X2 and P2X3 receptor immunoreactivity (Fig. 1), as were all HEK 293 cells stably expressing the heteromeric P2X2/3 channel (Kawashima et al. 1998). No immunostaining was observed in any of these cells when a P2X1-specific polyclonal antibody (Vulchanova et al. 1996) was used. As previously reported, P2X2-expressing HEK cells all showed immunoreactivity for the P2X2, but not P2X3, receptor while the converse was true for cells stably expressing the P2X3 receptor (Vulchanova et al. 1996, 1997; Kawashima et al. 1998).

Figure 1. P2X2 (red) and P2X3 (green) immunoreactivity in cultured rat nodose neurone.

Figure 1

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Preparation was double-labelled for P2X2 and P2X3 as described in Methods. Scale bar represents 25 μm.

ATP, but not α,β-meATP, activates P2X2 receptors while both of these agonists activate P2X3, P2X2/3 and P2X receptors on nodose neurones (see Evans & Surprenant, 1996). Therefore, in all experiments ATP was used as the agonist at P2X2-expressing cells but α,β-meATP was used for experiments on nodose neurones and on cells expressing P2X2/3 heteromeric receptors; this reduces the possibility that any homomeric P2X2 receptors, if present, may be contributing to the response. Peak amplitudes of currents in response to the first application of a maximally effective concentration of ATP or α,β-meATP (30 μM) were not significantly different among cells; these were 2.0 ± 0.3 nA (n = 36) in P2X2-expressing cells, 2.1 ± 0.2 nA (n = 37) in P2X3-expressing cells, 2.4 ± 0.3 nA (n = 36) for heteromeric P2X2/3 cells and 2.4 ± 0.25 nA (n = 39) in cultured nodose neurones. The rise times of these currents (measured as 10-90 % peak amplitude) were the same for nodose neurones and cells expressing the P2X2/3 heteromeric receptor (46 ± 5 ms and 48 ± 4 ms respectively) but were significantly greater for P2X2-expressing cells (82 ± 5 ms). We could not obtain reliable rise times for the rapidly desensitizing P2X3-induced currents because they were all faster than the equilibration time of the U-tube delivery system (10-15 ms). Desensitization during a 2 s application of agonist (30 μM) was quite variable from cell to cell in nodose neurones as well as in P2X2-expressing and P2X2/3-expressing cells. The desensitization, measured as the percentage change from peak amplitude at 2 s, ranged from 0 to 56 % (16 ± 3 %, n = 22) in nodose neurones, from 0 to 39 % (16 ± 2 %, n = 24) at cells expressing homomeric P2X2 receptors, and from 0 to 48 % (18 ± 3 %, n = 20) at cells expressing the heteromeric P2X2/3 receptor. Desensitization of the P2X3-induced current during the 2 s application period (to 30 μM ATP) was generally best fitted by the sum of two exponentials with time constants of 108 ± 11 ms and 1300 ± 169 ms (n = 18); these values are not significantly different from those reported previously from HEK 293 cells transiently transfected with P2X3 cDNA (Lewis et al. 1995).

Calcium permeability

Figures 2 and 3 show results from experiments designed to study the calcium permeability of P2X receptors by measuring reversal potentials in NMDG-containing external solution compared with similar measurements when 5 mM calcium was added to the NMDG solution. Mean reversal potentials and permeability ratios obtained from all such experiments are given in Table 1. Reversal potentials with bi-ionic NMDGo-Nai+ were the same for P2X2, P2X2/3 and nodose neurones (approximately −82 mV) but were significantly more positive for the P2X3 receptor (−66 mV), thus providing a PNMDG/PNa ratio for P2X3 receptors that is 2-fold greater than for the former receptors (Table 1). Taking into account this difference in PNMDG/PNa for the P2X3 receptor, the calculated PCa/PNa ratios for P2X3, P2X2/3 and nodose neurones are not significantly different (about 1.3) while the P2X2 receptor shows a 2-fold greater PCa/PNa ratio (Table 1).

Figure 2. Determination of purinoceptor-gated reversal potentials.

Figure 2

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Reversal potentials were determined for P2X2 (a), P2X3 (B), and P2X2/3 receptors (C) expressed in HEK 293 cells and P2X receptors in cultured nodose neurones (D) in control solution, in bi-ionic NMDG solution and after addition of 5 mM calcium to the NMDG solution (left to right as indicated). Recordings are from a single cell in each case. Representative holding potentials (mV) are indicated to the left of each set of superimposed currents. Bars above traces indicate duration of agonist application. Note ATP concentration was increased from 30 μM in control solution to 300 μM in NMDG and NMDG-Ca2+ in experiments on P2X2-transfected cells because of the strong calcium block of this receptor.

Figure 3. Current-voltage curves for experiments shown in Fig. 2.

Figure 3

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Current-voltage curves are shown for P2X2 (a), P2X3 (B) and P2X2/3 receptors (C), and P2X receptors in nodose neurones (D). In each graph the symbols used are as follows: •, control; ○, NMDG; ▪, NMDG + 5 mM calcium. Points are results from the entire range of holding potentials examined, which in some cases are greater than the number of traces shown in Fig. 2.

Table 1.

Reversal potentials (Vrev) and permeability ratios determined for P2X2, P2X3 and P2X⅔ receptors expressed in HEK 293 cells and for α,β-meATP-induced currents in nodose neurones

Vrev(NMDG) (mV) PNMDG/PNa Vrev(Ca2+) (mV) PCa/PNa
P2X2 −85 ± 2 (21) 0.03 ± 0.001 −49 ± 1 (14) 2.5 *
P2X3 −66 ± 1.3 (6) * 0.07 ± 0.003 * −51 ± 0.4 (6) 1.2
P2X⅔ −82 ± 2.5 (9) 0.04 ± 0.004 −57 ± 1.5 (10) 1.3
Nodose −83 ± 1.6 (29) 0.04 ± 0.002 −55 ± 1 (31) 1.5
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PCa/PNa is corrected for ionic activities (see Methods). Numbers in parentheses are numbers of cells.

*

Significantly different values from all other receptors (P < 0.001).

Calcium inhibition of P2X receptor currents

P2X2 receptors, but not P2X1 receptors, are strongly inhibited by external calcium (Evans et al. 1996). Therefore, we asked whether calcium may exert a similar differential inhibition at P2X3, P2X2/3 and nodose neurone P2X receptors. The inhibition by calcium of currents through the four receptors is shown in Fig. 4. In agreement with our previous study (Evans et al. 1996), the ATP-induced current in P2X2-expressing cells was quite sensitive to inhibition by external calcium (IC50 = 4.6 ± 0.2 mM, n = 6; Fig. 5). In contrast, the ATP or α,β-meATP-induced current in P2X3-expressing cells was relatively insensitive to blockade by external calcium (IC50 = 89 ± 4 mM, n = 5). The α,β-meATP-evoked currents in both nodose neurones and cells expressing the P2X2/3 heteromeric receptor showed an inhibition by calcium which was intermediate between that at the P2X2 and the P2X3 receptor (IC50 = 13.5 ± 1.2 mM (n = 5) and 15.4 ± 0.6 mM (n = 6), respectively). These values were not significantly different from each other but were significantly different from values at the P2X2 and P2X3 receptor (Fig. 5). We also asked whether the intermediate curves observed with the heteromeric channel and nodose neurones could be fitted by any proportional addition of the curves at the homomeric P2X2 and P2X3 receptor; data from the heteromeric and nodose neurones could not be fitted by any of our simulated curves using proportions from 1:20 to 20:1 in 0.05 steps.

Figure 4. Calcium block of purinoceptor-gated currents through heterologously expressed P2X2 (A), P2X3 (B), P2X2/3 (C) receptors and P2X receptors in nodose neurones (D).

Figure 4

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External calcium concentrations are indicated to the left of each series of superimposed currents; bars above traces indicate duration of agonist (30 μM throughout) application.

Figure 5. Summary of calcium inhibition of P2X2, P2X3, P2X2/3 and nodose neurone P2X receptors.

Figure 5

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A, concentration-inhibition curves from all results obtained in experiments as illustrated in Fig. 4 for P2X2 (•), P2X3 (▪), P2X2/3 (▴) and nodose neurones (♦). Lines are least squares fits to the Hill equation; each point is the mean ± s.e.m. of 4-6 experiments. IC50 values are (mM): 4.6 ± 0.2 (n = 6) for P2X2, 87 ± 4 (n = 4) for P2X3, 15.4 ± 0.6 (n = 6) for P2X2/3 and 13.5 ± 1.2 (n = 5) for nodose neurone P2X receptors, where the latter two values are not significantly different (P > 0.1) but are significantly different from the former two (P < 0.0001). B, α,β-meATP concentration-response curves for nodose neurones (•, ○) and P2X2/3 cells (▪, □), as indicated, in 2 mM Ca2+ (filled symbols) and 30 mM Ca2+ (open symbols). Data are means ± s.e.m. of 4 experiments.

The functional inhibition by external calcium (30 mM) of the α,β-meATP-induced current in nodose neurones and in cells expressing the P2X2/3 receptor was not different at −60, -20, 30 and 60 mV (n = 4). Calcium shifted the agonist concentration-response curves to the right with no depression of the maximum response at both nodose P2X receptors and P2X2/3 receptors, and the shift was the same for both receptors (Fig. 5B).

DISCUSSION

This study has demonstrated clear differences in calcium permeability and calcium block among rat P2X2, P2X3 and P2X2/3 receptors; for both sets of experiments results from nodose neurones closely resemble those of the P2X2/3 heteromeric receptor. The cultured nodose neurones used in the present study were also immunoreactive for both P2X2 and P2X3, but not P2X1, receptors. Thus, these results continue to support our previous suggestion that α,β-meATP-sensitive P2X receptors in nodose neurones comprise the heteromeric P2X2/3 subtype (Lewis et al. 1995). It should be emphasized that the experiments on nodose neurones (and HEK cells cotransfected with P2X2 and P2X3 cDNA) were designed to ‘isolate’ the heteromeric receptor by using only α,β-meATP, which is inactive at homomeric P2X2,4,5,6,7 receptors, as the agonist. Rat nodose neurones in situ express mRNA for P2X1-6 receptors (Collo et al. 1996) and the physiological transmitter is ATP which will activate all combinations of receptor subtypes. If multiple subtypes of P2X receptors were to be simultaneously activated in a single neurone, the differential Ca2+ permeability and block of P2X receptors demonstrated in the present study might be expected to result in a considerably variable Ca2+ influx and/or Ca2+ block of ATP currents depending upon the proportions of P2X receptor subunits expressed in any given neurone.

The calcium permeability of the P2X2 receptor is 2.5 times that of sodium, when measured with 5 mM calcium added to the external solution. This result confirms the observations of Evans et al. (1996). In contrast, the calcium permeability of the P2X3 receptor was about half this value (Table 1). Both of these are considerably less than that seen for the P2X1 receptor (PCa/PNa of 5 in 5 mM calcium and 3.9 in 112 mM calcium: Evans et al. 1996) or the P2X4 receptor (PCa/PNa of 4.2 in 112 mM calcium: Buell et al. 1996 or 4.2 in 8 mM calcium, assuming NMDG to be impermeant: Soto, Garcia-Guzman, Gomez-Hernandez, Hollman, Hkarshci & Stuhmer, 1996). Thus, the four homomeric receptors can be ranked with regard to calcium permeability as P2X1 ≈ P2X4 > P2X2 > P2X3. Cysteine-scanning mutagenesis has identified some residues which contribute to the aqueous lining of the pore of the P2X2 receptor; they are in or close to the second hydrophobic domain (Rassendren, Buell, Newbolt, North & Surprenant, 1997). A comparison of the differences among the receptors in calcium permeability with their differences in primary structure in this region might allow the identification of residues involved in binding permeating calcium ions, as has been done for other channels (reviewed by Sather, Yang & Tsien, 1994).

The inhibition by calcium of currents at P2X2 receptors was profound, with half-maximal block of the current observed at about 5 mM calcium (see also Evans et al. 1996). P2X3 receptors were more than 10-fold less sensitive (Fig. 5), although more sensitive to inhibition than currents elicited in cells expressing P2X1 receptors (Evans et al. 1996). Thus, the rank order for inhibition by calcium at the homomeric expressed receptors is P2X2 >> P2X3 > P2X1. Calcium block of the ATP current at the P2X2/3 heteromer and at the nodose neurone were voltage independent and were overcome by increasing the agonist concentration. We have previously obtained similar results for the calcium inhibition of the P2X2 homomeric channel (Evans et al. 1996). Such data are most amenable to explanation by a mechanism involving an allosteric alteration of the agonist binding site, rather than with a direct blockade of the ion channel itself (see also discussion by Evans et al. 1996). Detailed characterization of the mechanism(s) underlying Ca2+ block of P2X receptor subtypes will require single-channel data, which has not been pursued in the present study as the main aim was to distinguish similarities and differences between native P2X receptors expressed in nodose neurones and heterologously expressed P2X receptor subtypes. However, single-channel recordings of ATP-activated channels in phaeochromocytoma cells (in which ATP currents resemble homomeric P2X2 receptor responses) have shown that the Ca2+ block can be partially accounted for by a direct reduction in Na+ permeation (Nakazawa & Hess, 1993).

The inhibition at the P2X2/3 heteromer or nodose neurones was intermediate (IC50 ≈ 15 mM). This is the first functional property examined in the heteromeric channel in which neither the P2X2 phenotype, nor the P2X3 phenotype, was observed to be dominant. That is, the present and previous studies (Lewis et al. 1995) have shown that the kinetics of the P2X2/3 channel differ little from the P2X2 homomeric channel, while the pharmacology (i.e. α,β-meATP dose- response curve) does not differ significantly from the P2X3 channel. A strong differential sensitivity to pH at the P2X2 and P2X3 receptors also has been observed, with ATP-gated currents at the P2X2 receptor showing dramatic enhancement in lowered pH and the P2X3 receptor showing the converse (Stoop, Surprenant & North, 1997). The pH dependence of the P2X2/3 heteromeric channel is identical to that of the P2X2 receptor (Stoop et al. 1997), as is the ATP-gated current in nodose neurones (Li, Peoples & Weight, 1996). To date then, agonist kinetics and pH sensitivity of the heteromeric channel appear to be conferred primarily or solely by the P2X2 subunit, while calcium permeability and α,β-meATP sensitivity seem to be conferred by the P2X3 receptor. It will not be possible to attach any significance, or attempt any interpretation, of this intermediate calcium block in terms of protein structure until data concerning the stoichiometry of P2X receptors become available. Nevertheless, it seems clear that both subunits in the heteromeric channel must contribute to the calcium binding site(s) involved in inhibition of the ATP current.

P2X3 receptors are uniquely localized to a subset of nociceptive neurones in sensory ganglia (Chen et al. 1995; Vulchanova et al. 1997; Cook et al. 1997); thus, they may be expected to play a role in peripheral and central pain pathways when expressed either as homomeric or heteromeric receptors. All of the neurones in our cultures of rat nodose neurones were immunoreactive for both P2X2 and P2X3 receptors and all displayed a similar response to α,β-meATP. Thus, α,β-meATP responses in nodose neurones are indistinguishable from responses at P2X2/3 heteromeric receptors expressed in HEK 293 cells in terms of their α,β-meATP concentration-response relation, kinetics, pH sensitivity, calcium permeability and calcium block. It is particularly surprising that all neurones which survive under our culture conditions (and others, see Li, Peoples & Weight, 1996) are immunoreactive for P2X3 and show α,β-meATP-evoked currents in view of recent findings that the subpopulation of nociceptor neurones that express P2X3 receptor protein also selectively bind the isolectin IB4 (Vulchanova et al. 1996) and that glial cell line-derived neurotrophic factor (GDNF), but not NGF, supports the survivial of IB4-binding nociceptive DRG neurones in culture (Molliver et al. 1997). We used NGF to culture nodose neurones; thus, P2X3-containing neurones may have been expected not to have survived. Further studies, in which the survival and/or differentiation of P2X3/IB4-positive nodose neurones are followed during in vitro culture procedures using NGF or GDNF, will be required to clarify the present discrepancy. Nevertheless, we have now identified a further distinct phenotype of the heteromeric P2X2/3 channel: its block by external calcium, that can be used to distinguish the native heteromeric channel in neurones, especially in other sensory neurones. In the case of calcium permeability, the physiological implications may be considerable. For example, sensory neurones likely to subserve nociception because they innervate the tooth pulp exhibit two kinds of response to α,β-meATP (Cook et al. 1997). These include a transient component probably mediated by homomeric P2X3 receptors, and a sustained component resembling that seen in our nodose ganglion cells which could be mediated by P2X2/3 receptors. The present work indicates that neither of these species has a high calcium permeability when compared with some other P2X receptors.

Acknowledgments

We thank Daniele Estoppey and Denis Fahmi for cell culture and immunohistochemistry. Bob Elde (University of Minnesota) generously supplied the P2X polyclonal antibodies.

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