The Journal of Neurophysiology Vol. 78 No. 4 October 1997, pp. 1837-1840
Copyright ©1997 by the American Physiological Society

Different Sensitivities to pH of ATP-Induced Currents at Four Cloned P2X Receptors

Ron Stoop, Annmarie Surprenant, and R. Alan North

Geneva Biomedical Research Institute, Glaxo Wellcome Research and Development, Plan-les-Ouates, 1228 Geneva, Switzerland

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Stoop, Ron, Annmarie Surprenant, and R. Alan North. Different sensitivities to pH of ATP-induced currents at four cloned P2X receptors. J. Neurophysiol. 78: 1837-1840, 1997. The effect of changing extracellular pH was studied on the currents induced by ATP or -methylene-ATP in HEK293 cells transfected with different P2X receptor subunits. In cells expressing P2X₁, P2X₃, or P2X₄ receptors, the effect of ATP was decreased by acidification. In cells expressing P2X₂ receptors, acidification increased the ATP-induced current; this effect was also seen in cells expressing heteromeric P2X₂ and P2X₃ receptors. At P2X₂ receptors, acidification caused a leftward shift in the ATP concentration-response curve, without change in maximum; the pK_a for this effect was 7.3. At P2X₄ receptors, acidification caused a rightward shift in the ATP concentration-response curve, without change in the maximum; the pK_a for this effect was 6.8. The pH dependence of the action of ATP should be taken into account in studies of synaptic transmission, and it may provide a further tool to assign molecular identity to P2X receptors expressed by brain neurons.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Changes in extracellular pH can be particularly marked in the limited extracellular space of the nervous system, where they can result from intense neuronal activity (Chesler and Kaila 1992). Such changes may influence the function of cell surface molecules such as receptors and ion channels. For example, currents activated by glutamate acting at N-methyl-D-aspartate receptors are decreased by acidification (Tang et al. 1990; Traynelis and Cull-Candy 1990), whereas the opposite effect is seen for the action of -aminobutyric acid (GABA) at GABA_A receptors (Pasternak et al. 1992). The proton sensitivity of the GABA response depends on the subunit composition of the receptor (Krishek et al. 1996); in the case of the neuromuscular acetylcholine nicotinic receptor, it depends on the species (Li and McNamee 1992).

P2X receptors for ATP receptors are also pH sensitive. Protons strongly potentiate the effect of ATP in rat nodose ganglion neurons (Li et al. 1996). A similar effect has been reported for cloned P2X₂ receptors expressed in Xenopus oocytes (King et al. 1996). However, there is a family of P2X receptor subunits (Collo et al. 1996), and it seemed worthwhile to study the effects of pH on further subunits for several reasons. First, the most abundant subunit RNAs found in the CNS are P2X₄ and P2X₆ rather than P2X₂ (Collo et al. 1996); P2X₂ receptor subunits have a rather limited central distribution (Vulchanova et al. 1996). Second, currently available pharmacological methods to discriminate among P2X receptor subtypes are limited; they are desensitization and effectiveness of the agonist -methylene-ATP and the antagonist pyridoxal-5-phosphate-6-azophenyl-2,4-disulfonic acid (see Collo et al. 1996); differences in pH sensitivity among subunits could complement these. More specifically, channels in native membrane can form as heteromultimers, and this seems to occur particularly in sensory ganglia such as the nodose ganglion (Lewis et al. 1995); it was therefore important to determine the pH sensitivity of the heteromultimeric receptors in addition to the homomultimeric receptors. Finally, it is possible, perhaps likely, that differences in pH sensitivity among the different receptors might result from differences in titratable amino acid side chains on the exposed extracellular aspect of the channel, and their identification could provide useful clues as to the disposition of the protein within the membrane. For these reasons, we investigated the effects of changing pH on currents induced by ATP in mammalian cells expressing P2X₁, P2X₂, P2X₃, and P2X₄ receptors as well as cells expressing a heteromeric combination of P2X₂ and P2X₃ subunits (P2X_2/3).

	METHODS

Abstract Introduction Methods Results Discussion References

Heterologous expression of P2X receptors

cDNA encoding the P2X₁, P2X₂, P2X₃, and P2X₄ receptors was originally cloned from human bladder smooth muscle (Valera et al. 1995), rat pheochromocytoma cells (Brake et al. 1994) (gift of D. Julius, University of California at San Francisco, CA), rat dorsal root ganglion cells (Lewis et al. 1995), and rat brain (Buell et al. 1996), respectively. All were subcloned in pCDNA3 (Stratagene).

For transient transfections, 1 ml of Optimem (Life Technologies) containing 1 µg of cDNA and 5 µg of lipofectin were placed in a 35-mm Petri dish containing four coverslips on which human embryonic kidney (HEK) 293 cells were plated (5 × 10³ cells per coverslip). This medium was removed after 5-6 h of incubation at 37°C and replaced with normal culture medium; recordings were made 12-48 h later. More than 90% of the cells from which recordings were made responded to ATP, whereas untransfected cells did not (see Evans et al. 1995). Stably transfected HEK 293 cells expressing P2X₁ receptors, P2X₂ receptors, and P2X_2/3 receptors were also used (Evans et al. 1995; Kawashima et al. 1997).

Semliki forest virus was also used for expression of human P2X₁ and rat P2X₂, P2X₃, and P2X₄ receptors in Chinese hamster ovary (CHO) cells; the methods have been fully described in Evans et al. (1996). Experiments with transfected HEK 293 cells and infected CHO cells gave similar results for the individual P2X receptors and the experimental results obtained with both cell types have therefore been pooled.

Electrophysiological recording

Whole cell recordings were made from HEK 293 cells with the use of an Axopatch 200 patch-clamp amplifier (Axon Instruments). Patch pipettes (4-7 M) contained (in mM) 145 potassium aspartate, 11 ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid, 5 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), and 5 NaCl; the external solution contained 145 NaCl, 2 KCl, 1 MgCl₂, 2.5 CaCl₂, 10 HEPES, and 10 mM glucose. Agonists were applied with the use of a fast-flow U-tube delivery system (Fenwick et al. 1982), whereas pH was changed in both the superfusate and the U-tube solution that contained the agonist. The changes in pH themselves did not cause significant changes in holding current at 60 mV. Agonist concentration-response curves were constructed by expressing currents as percentages of the maximal current evoked by the agonist at pH 7.3 in HEK or CHO cells. All currents were recorded at a holding potential of 60 mV. P2X₁ and P2X₃ receptors exhibit strong desensitization (Surprenant et al. 1995; Valera et al. 1995). Reproducible responses were obtained with these receptors by applying agonist for 1-s at intervals of 5 min. P2X₂, P2X_2/3, and P2X₄ receptors exhibit little or no desensitization and agonists were applied to these receptors for similar durations but at intervals of 60 s. Concentration-response curves were fitted by hyperbolic functions with the use of the least-squares method. ATP disodium salt, -methylene-ATP lithium salt (meATP) and diethylpyrocarbonate were obtained from Sigma. Concentrations of the various forms of ATP were calculated with the use of EqCal (Biosoft).

	RESULTS

Abstract Introduction Methods Results Discussion References

Acidification had quite opposite effects on the ATP-induced currents in cells expressing P2X₁, P2X₃, and P2X₄receptors as compared with cells expressing P2X₂ or P2X_2/3receptors. Figure 1 illustrates this for the five sets of cells for currents evoked by ATP or meATP applied at concentrations giving half-maximal effects (see Collo et al. 1996). Figure 2 summarizes the results of all such experiments. In all cases, the effects of pH change reached a steady level within 30-60 s after the change in extracellular pH and did not alter during longer exposure (up to 5 min).

View larger version (18K): [in this window] [in a new window]   FIG. 1. Inward currents in response to activation of P2X receptors expressed in HEK293 or CHO cells. Each set of 3 traces shows recordings from 1 cell at different pHs. Horizontal bars: application time of agonists at the concentrations indicated.

View larger version (30K): [in this window] [in a new window]   FIG. 2. Mean peak currents evoked by ATP or -methylene-ATP lithium salt (meATP) at pH 6.3, 7.3, and 8.3. Currents are normalized to the value at 7.3 in the same cell. Agonist, concentration, and number of cells tested were as follows: P2X1: ATP 1 µM, n = 4; P2X3: meATP1 µM, n = 4; P2X4: ATP 10 µM, n = 6; P2X2: ATP 10 µM, n = 7; P2X2/3;meATP 1 µM, n = 3. Vertical bars: means ± SE.

For P2X₁, P2X₃, and P2X₄ receptors, currents were decreased by acidification (pH 6.3) but little changed by alkalinization (pH 8.3). Currents at P2X₁ and P2X₃ receptors desensitize strongly during agonist applications lasting for 1 s (Fig. 1) (see Lewis et al. 1995; Valera et al. 1995), but the rate of desensitization was not obviously affected by the changes in pH (Fig. 1). For P2X₂ (and P2X_2/3) receptors, acidification to pH 6.3 clearly increased the current, but alkalinization also caused a strong inhibition. A direct comparison between P2X₂ and P2X₄ receptors is shown in Fig. 3, in which the pH was varied systematically over a wider range. The P2X₂ receptor behaved as though it had a single titratable site responsible for changing the current amplitude with a pK_a of 7.3. In contrast, currents at the P2X₄ receptor responded in the opposite direction to changes in pH with a pK_a of ~6.8.

View larger version (31K): [in this window] [in a new window]   FIG. 3. Concentration-response curves for ATP at P2X2 (top left) and P2X4 (bottom left) receptors for pH 6.3 (), 7.3 (hatched squares), and 8.3 (). Titration curves generated for 10 µM ATP for P2X2 (top right) and P2X4 (bottom right) receptors. Concentration-response values were normalized to the maximal response at pH 7.3 and show means ± SE (n = 3-6). Arrowheads: pH 6.3, 7.3, and 8.3. Lines are fitted to logistic functions. pH titration curves show means ± SE for 13 (P2X2) or 8 (P2X4) experiments normalized to the responses obtained at pH 7.3. Lines: best fits to logistic functions constrained to unit coefficient.

The effects of pH on cells expressing the P2X₂ and P2X₄ receptors were studied over a range of ATP concentrations. In each case, there was no effect of pH on the maximal current induced by ATP and no marked effect on the slope of the concentration-response curves (Fig. 3). Thus the main effect of pH in each case was a parallel shift in the ATP concentration-response curve. Alteration in pH by two log units (6.3-8.3) was equivalent to a 30- to 100-fold change in ATP concentration; the respective pEC₅₀ values (negative logarithm of concentration causing half-maximal effect) for pH 6.3, 7.3, and 8.3 were 5.4, 4.8, and 3.7 in the case of P2X₂ receptors and 5.7, 5.3, and 4.5 for P2X₄ receptors (Fig. 3).

The pK_a values for the P2X₂ and P2X₄ receptors are closest to those of histidine. Diethylpyrocarbonate, which carbethoxylates the imidazole ring of histidine (as well as the side groups of arginine, tyrosine, and cysteine) (Leonard et al. 1970), did not change the effects of acidification on ATP-evoked currents at P2X₂ or P2X₄ receptors (Fig. 4). This treatment (diethylpyrocarbonate, 500 µM, 3 min, pH 6.3) reduced the maximal currents evoked by ATP to ~50% of their control values in the case of the P2X₂ receptor (Fig. 4), but it did not change the maximal current in the case of P2X₄ receptors (n = 6).

View larger version (22K): [in this window] [in a new window]   FIG. 4. Diethylpyrocarbonate does not prevent the effect of pH at P2X2 receptors. Concentration-response curves for ATP at P2X2 receptors before (, ) and after (, ) application of diethylpyrocarbonate (500 µM; 3 min). In both conditions, acidification caused a leftward shift in the concentration-response curve. Values are means ± SE from 4-6 experiments and are normalized to the maximum response obtained at pH 7.3 before diethylpyrocarbonate treatment. Lines: best fits to logistic functions. pEC50 values at pH 6.3 and 7.3: 5.3 and 4.8 for P2X2 receptors and 5.4 and 4.1 for P2X4 receptors, respectively.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

The changes in current induced by ATP observed in the present experiments occurred rapidly and reversed rapidly when the control pH was restored. We are therefore inclined to interpret them as resulting from changes occurring outside the cell rather than within the cell. In separate experiments, it was shown that changes in intracellular pH (6-9) do not lead to significant changes in the action of ATP at P2X₂ receptors (C. Lewis and A. Surprenant, unpublished observations). The lack of effect of pH changes on the maximum current amplitude is more consistent with a change in the form of the ligand or its binding site, rather than a change in the conducting pore, although single-channel studies or permeability measurements would be needed to address the latter possibility. There are two reasons for thinking that the changes in current do not result from changes in the form of the ATP itself. First, currents are increased at some receptors and decreased at others. Second, calculations of the concentrations of ATP⁴, MgATP², and CaATP² in the solutions used indicate that they change by <10% when pH is altered in the range of 6.3-9.3 (see also Li et al. 1996).

The contrasting sensitivity to pH of the P2X₂ and P2X₄ receptors therefore seems likely to result directly from differences in amino acid composition of the proteins. The four receptors all have histidine residues within their presumed extracellular loops (see Collo et al. 1996), but they are not at all conserved. In this loop, the P2X₁ receptor has 10 histidines, the P2X₂ receptor 9, the P2X₃ receptor 2, and the P2X₄ receptor 3, but only 1 of these residues occurs in an equivalent position in only one of the pairwise alignments of the proteins. For example, there is no histidine that is common to P2X₁, P2X₃, and P2X₄ but missing from P2X₂ and that might therefore account for the differences observed. It was possible that protonation of a histidine residue in a position unique to the P2X₂ receptor was responsible for the increase in current at low pH observed uniquely for that receptor, but this also seems unlikely in view of the lack of any effect of diethylpyrocarbonate. Further experiments will be needed to elucidate the particular amino acids that are responsible for the pH effects; histidines plays critical role in the binding of ATP to a P2Y receptor (Erb et al. 1995).

The cells stably expressing P2X_2/3 receptors gave a relatively sustained response to meATP, indicating that the receptor population activated contained heteromeric complexes (see Kawashima et al. 1997; Lewis et al. 1995). These had the same sensitivity to pH as homomeric P2X₂ receptors, being increased by acidification and decreased by alkalinization. This provides a further phenotype (pH sensitivity) that is brought by the P2X₂ subunits to the heteromeric assembly, in addition to the kinetic properties (slower activation, little or no decline during the application, and lack of "rundown" with repeated applications) (see Lewis et al. 1996). It is noteworthy that nodose ganglion cells show the same effect of pH (Li et al. 1996) as observed here for the P2X_2/3 heteromer, which is further evidence consistent with the view that their receptors mostly comprise P2X_2/3 heteromers (Lewis et al. 1995).

The physiological significance of the pH dependence reported here remains to be assessed. The inhibition of P2X₄ receptor-mediated currents observed by acidification (Fig. 3) suggests that a change in pH of one-half unit would profoundly reduce the effectiveness of ATP. If ATP is coreleased with glutamate at central synapses, any concomitant acidification would reduce its effects. Similarly, changes in pH arising from neuronal activity or other causes (Chesler and Kaila 1992) would alter the effectiveness of ATP. It will be important to assess these effects by examining directly the effect of pH on ATP-mediated synaptic transmission.

	ACKNOWLEDGEMENTS

  We thank D. Estoppey for cell culture and transfections.

	FOOTNOTES

  Address reprint requests to R. A. North.

  Received 26 February 1997; accepted in final form 12 June 1997.

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