Abstract
There are currently seven P2X receptor subunits (P2X1–7) defined by molecular cloning. The functional identification of these receptors has relied primarily on the potency of α,β-methylene-ATP relative to that of ATP and on the kinetics of receptor desensitization. In the present experiments we found that the 2′,3′-O-(2,4,6-trinitrophenyl)-substituted analogs of ATP are selective and potent antagonists at some but not all P2X receptors. The trinitrophenyl analogs of ATP, ADP, AMP, and GTP produced a reversible inhibition of ATP-evoked currents in human embryonic kidney 293 cells expressing P2X1 receptors, P2X3 receptors, or both P2X2 and P2X3 (heteromeric) receptors; IC50 values were close to 1 nm. These compounds were at least 1000-fold less effective in blocking currents in cells expressing P2X2, P2X4, or P2X7receptors (P2X5 and P2X6 not tested). GTP, 2,4,6-trinitrophenol, and the 2′,3′-trinitrophenyl analog of adenosine (0.1–10 μm) had no effect. Thus, we have identified a structural motif that confers antagonist action at P2X receptors that contain P2X1 or P2X3 subunits (the α,β-methylene-ATP-sensitive subclass).
There are seven P2X receptor subunits, which assemble into ATP-activated ion channels either as homomers or heteromers (reviewed by North, 1996;North and Barnard, 1997). At the molecular level, any pair of the subunits has 35–50% identical amino acids. At the functional level, several subgroups have been distinguished. For example, in one subgroup (P2X1 and P2X3 homomeric channels), αβmeATP and ATP are equally effective agonists, and the currents desensitize during agonist applications of more than several hundred milliseconds. None of the other homomeric channels is activated by αβmeATP, and the currents show much less desensitization. A distinct class of channel is formed by the coexpression of P2X2 and P2X3 subunits; this class is activated by αβmeATP and ATP but it shows little desensitization. A further distinguishing feature is the ability of PPADS to block the currents evoked by ATP; P2X4, P2X6, and P2X7 receptors are relatively insensitive. Finally, P2X7homomeric channels are fundamentally different from all the others because repeated or prolonged agonist application results in cell permeabilization as measured by the uptake of fluorescent dyes and, eventually, cell lysis (North, 1996; Surprenant et al., 1996; North and Barnard, 1997).
The assignment of functional roles for P2X receptors in intact tissues depends critically on the use of receptor antagonists. Indeed, the main evidence that ATP mediates synaptic transmission between neurons (Edwards et al., 1992; Evans et al., 1992) or from nerve to muscle (Sneddon and Westfall, 1984; Evans and Surprenant, 1992) has been the block of the postsynaptic responses by suramin and/or PPADS (Sneddon and Westfall, 1984; Dunn and Blakeley, 1988;Ziganshin et al., 1994). However, the low affinity and limited specificity of these compounds restricts their usefulness and, as mentioned above, some P2X receptors are not blocked (Buell et al., 1996). There is a clear need to identify more receptor antagonists.
Trinitrophenyl analogs of ATP have been widely used for the fluorescent labeling of ATP binding sites in proteins, including P2X receptors (Mockett et al., 1994). We first examined their effects on cloned and expressed P2X receptors with such an application in mind. In the course of those experiments, it became clear that, for some P2X receptors, the analogs were able to block responses to ATP at nanomolar concentrations. Here we report the characterization of this observation.
Experimental Procedures
HEK 293 cells that stably or transiently express the following P2X receptors were used in these studies: human P2X1, rat P2X2, rat P2X3, rat or human P2X4, rat P2X2 together with rat P2X3 (heteromer), and rat P2X7. Generation of stable P2X receptor-expressing cell lines and methods of transient lipofectin transfection have been described in detail previously (Evans et al., 1995; Buell et al., 1996; Evans et al., 1996; Kawashima et al., 1997). HEK cells stably transfected with the human P2X4 receptor were generously provided by Professor W. Stuhmer, Max-Planck Institute (Gottingen, Germany). Cells were plated onto 12-mm glass coverslips and maintained in Dulbecco’s modified Eagle’s medium, Nutrient Mix F-12 (GIBCO-BRL, Bethesda, MD) supplemented with 10% heat-inactivated fetal calf serum (FAKOLA, Bern, Switzerland) and 2 mml-glutamine at 37° in a humidified 5% CO2 incubator.
Whole-cell recordings were made 12–48 hr after transient transfection (rat P2X1, P2X3, P2X4) and 6–72 hr after passage of stable cell lines (human P2X1, P2X3, P2X4, and rat P2X2, P2X2/3, and P2X7). Currents were recorded with an EPC9 patch-clamp amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany), acquired (1–2 kHz) and analyzed with Pulse and PulseFit 8.02 (HEKA). Patch pipettes (4–7m[/omega]) contained 140 mm NaCl, 10 mm HEPES, and 11 mm EGTA. The external solution was 147 mm NaCl, 10 mm HEPES, 12 mm glucose, 2 mm KCl, 2 mmCaCl2, and 1 mmMgCl2. Osmolarity and pH values of both solutions were maintained at 300–315 mOsm/liter and 7.3, respectively. Unless otherwise stated, experiments were performed at a holding potential of −60 mV and at room temperature. Agonists were applied using a fast-flow U-tube delivery system (Fenwick et al., 1982). Antagonists were added to both the bath superfusate and the fast-flow solution. ATP was the agonist in all experiments on P2X1, P2X2, P2X4, and P2X7 receptors. Both ATP and αβmeATP were used at the P2X3receptor and only αβmeATP was used at the heteromeric P2X2/3 receptor (Kawashima et al., 1997). Agonists were applied for 0.5–2-sec duration at 2 min intervals at all receptors except P2X1 and P2X3 where 4–5 min intervals were required to allow recovery from desensitization (Evans et al., 1995).
Agonist concentration-response curves for each cell were fit by the least-squares method to I = Imax[1 + (EC50/[ A])nH] whereI is the peak current evoked by agonist concentration [A], Imax is the peak current evoked by a maximal agonist concentration, EC50is the concentration giving half the maximal current, andnH is the Hill coefficient. Antagonist concentration-inhibition curves were obtained in individual cells by using a fixed agonist concentration close to the EC50 (1 μm ATP at P2X1, 10 μm ATP at P2X2 and P2X4, 300 μm ATP at P2X7, 1 μm ATP or αβmeATP at P2X3, and 5 μmαβmeATP at P2X2/3), and progressively increasing the concentration of antagonist: IC50values were calculated by least squares fitting to I = I0/[1 + ( IC50/[ Ant])−nH] , where I and I0 are peak currents in the presence and absence of antagonist at concentration [Ant]. All EC50 and IC50 values given in text and tables are the mean ± standard error from individual cells; however, the graphs in the figures were drawn by averaging results from all experiments and fitting a single concentration-response curve to the pooled data.
TNP-AMP sodium salt, TNP-ADP disodium salt, TNP-ATP trisodium salt, and TNP-GTP trisodium salt were obtained from Molecular Probes (Eugene, OR). GTP, ATP, and αβmeATP were from Sigma (St. Louis, MO) and 2,4,6-trinitrophenol (picric acid) was from Fluka (Buchs, Switzerland). TNP-A was prepared from adenosine and 2,4,6-trinitrobenzenesulfonate according to the procedure published by Azegami and Iwai (1975); the red precipitate of TNP-A that crystallized from the solution was purified by reprecipitation from acetone (1 ml) by adding 10 volumes of toluene.
Results
TNP-ATP strongly inhibited currents in cells expressing P2X1, P2X3, or P2X2/3 receptors (IC50about 1 nm), but was very much less effective in cells expressing P2X2, P2X4, or P2X7 receptors (IC50 > 1 μm)(Figs. 1 and2; Table1). The inhibition was concentration-dependent and well fitted by the logistic function (see Experimental Procedures); the IC50 values are shown in Table 1, and the coefficient nHwas not significantly different from unity. The inhibition reversed within 4–15 min of TNP-ATP washout, although reversal was sometimes incomplete for near maximal concentrations. The inhibition was the same at holding potentials of −60 mV and 40 mV (n = 4, 6, 5, and 3 for cells expressing P2X3, P2X2/3, P2X2, and P2X4 receptors, respectively). The P2X7 receptor was particularly insensitive to blocking by TNP-ATP; at the highest concentration tested (30 μm), the inhibition was 39 ± 2% (n= 6). TNP-ATP (1 nm-30 μm) had no agonist action at any of the P2X receptors.
TNP-ATP is a potent antagonist at P2X1, P2X3, and P2X2/3 receptors. Each set of records consists of superimposed currents recorded from individual HEK 293 cells expressing the indicated receptor before, during, and after application of TNP-ATP at 10 nm (P2X1, P2X3, and P2X2/3) or at 30 μm(P2X2, P2X4, and P2X7). Currents shown in the presence of TNP-ATP are after 4-min application; currents shown after TNP-ATP are at 4-min wash except for P2X1, in which case the washout was for 8 min.
TNP-ATP concentration-inhibition curves generated from all experiments as illustrated in Fig. 1. Results are plotted as normalized current, where current in absence of antagonist is equal to 1. Points, mean ± standard error of 4–8 experiments. Lines, least-square fits to a logistic equation (see Experimental Procedures).
Inhibition of currents evoked by ATP or αβmeATP in cells expressing P2X receptor
The effect of TNP-ATP was mimicked by TNP-ADP and TNP-AMP, as well as TNP-GTP (Fig. 3), although TNP-A had no effect (n = 4). These compounds were also highly effective at P2X1, P2X3, and P2X2/3 receptors but much less so at P2X2, P2X4, and P2X7 receptors. Complete antagonist-inhibition curves were generated for P2X2, P2X3, and P2X2/3 receptors (Fig. 3), and IC50 values are provided in Table1. The dose-inhibition curves at the heteromeric P2X2/3 receptors were consistently to the right of those for the homomeric P2X3 receptor (Fig.3), although the difference in IC50 estimates was significant only in the case of TNP-ATP (Table 1). As for TNP-ATP, the other TNP-nucleotides (up to 30 μm) had no agonist action. TNP-A (0.1–1 μm), GTP (0.1–10 μm), and picric acid (10 μm) had no agonist or antagonist action at P2X3, P2X2/3, or P2X2 receptors.
TNP-AMP, TNP-ADP, and TNP-GTP are also potent antagonists at P2X3 and P2X2/3 receptors.Sets of traces, currents recorded from a single cell expressing P2X2 (left), P2X3(middle), or P2X2/3 (right) receptors in the absence and presence of increasing concentrations of TNP-GTP, as indicated. Graphs are concentration-inhibition curves obtained from all such experiments for TNP-AMP (left), TNP-ADP (middle), and TNP-GTP (right).Points, mean ± standard error of 4–8 experiments.
The nature of the inhibition was examined further in the case of the P2X3 receptor by constructing full agonist concentration-response curves. With either ATP or αβmeATP as the agonist, TNP-ATP (3 and 10 nm, respectively) caused both a rightward shift and a depression of the maximal current, indicating insurmountable antagonism. For the two antagonist concentrations ([B])(3 and 10 nm, respectively), the curves were fit by an expression appropriate to noncompetitive antagonism [I/Imax = [1 + (EC50/[A])]−1(1 + KB /[B])−1], which provided estimates of KB of about 2 nm. Similar results were obtained for TNP-ADP and TNP-GTP (data not shown).
Discussion
The results indicate that certain nucleotides with a ribose-substituted trinitrophenyl group are potent antagonists at those P2X receptors that can be activated by αβmeATP (P2X1, P2X3, and P2X2/3). However, several observations are not readily reconciled with the notion that the TNP-nucleotides are binding to the site occupied by the agonists when they act to open the P2X receptor channel. First, the antagonism is noncompetitive (Fig.4). Second, both guanine and adenine nucleotides are equally effective; this is in marked contrast to the lack of any agonist activity by GTP itself. Third, removal of one or even two phosphate groups from TNP-ATP had no significant effect on the antagonism; yet in terms of agonist action, ADP is more than 100-fold less potent than ATP at the P2X3 receptor (Lewiset al., 1995) and AMP (100 μm) has no effect at the P2X1 (Evans et al., 1995), P2X3 (Chen et al., 1995) or P2X2/3 receptor (unpublished observations). Removal of the third phosphate, as in TNP-A, resulted in loss of antagonism. In brief, the antagonist binding site differs from the agonist binding site in that it does not discriminate between guanine or adenine bases, absolutely requires the 2′,3′-trinitrophenol, and will accept one, two, or three (but not zero) 5′-phosphates.
TNP-ATP is a noncompetitive antagonist at the P2X3 receptor. a, Currents recorded from a single cell expressing the P2X3 receptor in the absence (top) and presence (bottom) of 10 nm TNP-ATP. b and c, Agonist concentration-response curves for αβmeATP (b) and ATP (c) in the absence and presence of 3 nm or 10 nm TNP-ATP. Points, mean ± standard error of 8–9 experiments for αβmeATP and 3–6 experiments for ATP.
It is possible that the TNP-nucleotides directly block the conducting pathway of the channel, as found for the outwardly rectifying chloride channel (Paulmichi et al., 1992; Venglarik et al., 1993). This seems unlikely both because ATP is negatively charged and the channel is cation-selective, and because the inhibition by TNP was not different for inward and outward currents. In any event the concentrations of extracellular ATP and TNP-ATP that block the outwardly rectifying chloride current are still some hundred-fold higher than those effective at P2X1, P2X3, and P2X2/3 receptors. The most likely mechanism, therefore, is the binding of TNP-nucleotides to an allosteric site on the large extracellular region of the receptor. In this case, the P2X1 and P2X3 subunits might provide a common domain that interacts with the strongly electronegative trinitrophenyl moiety. It is interesting that the most potent antagonists in a series tested on the rat urinary bladder (which expresses P2X1receptors) also had large aromatic 3′ substitutions (Bo et al., 1994; Burnstock et al., 1994); these bound with affinities in the 10–100 nm range.
The weak antagonism of TNP-ATP at other receptors had previously been reported for cochlear hair cells isolated from guinea pig organ of Corti (Mockett et al., 1994), which are known to express P2X2 receptors (Housley et al., 1995;Brandle et al., 1997). In that case, 75 μmTNP-ATP almost completely blocked the current evoked by 10 μm ATP. TNP-ATP is not an effective antagonist in the rat parotid gland (Soltoff et al., 1993), which contains P2X4 (Buell et al., 1996) and P2X7 receptors (Collo et al., 1997). It will clearly be important to test the TNP analogs on P2X responses to ATP in other tissues. On the basis of the present work, one might expect blockade in the nanomolar concentration range to indicate that the underlying receptor contains P2X1 or P2X3 subunits. The results with heteromeric receptors, such as are expressed by some primary afferent neurons (Lewis et al., 1995; Cook et al., 1997), might be less straightforward. In the present work, we used αβmeATP as the agonist in experiments on the cells expressing the heteromeric P2X2/3 receptor, on the assumption that it activates only heteromers and not any homomeric P2X2 receptors that might also be present. The nerve-released transmitter would be ATP rather than αβmeATP, and a combined action of P2X2 and P2X3 subunits might result in an intermediate sensitivity to TNP-ATP. For the interpretation of such experiments it would also be useful to know whether these TNP analogs have blocking action at other receptors types, including the P2Y receptors.
Acknowledgments
We thank D. Estoppey for her skilled assistance with cell culturing.
Footnotes
-
Send reprint requests to: Dr. R. A. North, Institute of Molecular Pharmacology, Department of Biomedical Sciences, University of Sheffield, Alfred Denny Building, Sheffield SIO 2TN, England, UK. E-mail: r.a.north{at}sheffield.ac.uk
-
↵1 Current affiliation: Department of Pharmacology, Glaxo Wellcome Research and Development, 37135 Verona, Italy
- Abbreviations:
- αβmeATP
- α,β-methylene-ATP
- HEK
- human embryonic kidney
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- PPADS
- pyridoxal 5-phosphate 6-azophenyl-2′,4′-disulphonic acid
- TNP
- trinitrophenyl
- TNP-A
- 2′,3′-O-(2,4,6-trinitrophenyl)-adenosine
- TNP-ADP
- 2′,3′-O-(2,4,6-trinitrophenyl)-ADP
- TNP-AMP
- 2′,3′-O-(2,4,6-trinitrophenyl)-AMP
- TNP-ATP
- 2′,3′-O-(2,4,6-trinitrophenyl)-ATP
- TNP-GTP
- 2′,3′-O-(2,4,6-trinitrophenyl)-GTP
- EGTA
- ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′,-tetraacetic acid
- Received January 12, 1998.
- Accepted February 18, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
