Trinitrophenyl-Substituted Nucleotides Are Potent Antagonists Selective for P 2 X 1 , P 2 X 3 , and Heteromeric P 2 X 2 / 3 Receptors

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 a,b-methyleneATP relative to that of ATP and on the kinetics of receptor desensitization. In the present experiments we found that the 29,39-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 P2X7 receptors (P2X5 and P2X6 not tested). GTP, 2,4,6-trinitrophenol, and the 29,39-trinitrophenyl analog of adenosine (0.1–10 mM) had no effect. Thus, we have identified a structural motif that confers antagonist action at P2X receptors that contain P2X1 or P2X3 subunits (the a,bmethylene-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), abmeATP 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 abmeATP, 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 abmeATP 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, P2X7 homomeric 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, 1 Current affiliation: Department of Pharmacology, Glaxo Wellcome Research and Development, 37135 Verona, Italy ABBREVIATIONS: abmeATP, a,b-methylene-ATP; HEK, human embryonic kidney; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; PPADS, pyridoxal 5-phosphate 6-azophenyl-29,49-disulphonic acid; TNP, trinitrophenyl; TNP-A, 29,39-O-(2,4,6-trinitrophenyl)-adenosine; TNPADP, 29,39-O-(2,4,6-trinitrophenyl)-ADP; TNP-AMP, 29,39-O-(2,4,6-trinitrophenyl)-AMP; TNP-ATP, 29,39-O-(2,4,6-trinitrophenyl)-ATP; TNP-GTP, 29,39-O-(2,4,6-trinitrophenyl)-GTP; EGTA, ethylene glycol bis(b-aminoethyl ether)-N,N,N9,N9,-tetraacetic acid. 0026-895X/98/060969-05$3.00/0 Copyright © by The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 53:969–973 (1998). 969 at A PE T Jornals on M ay 7, 2017 m oharm .aspeurnals.org D ow nladed from 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 (GIBCOBRL, Bethesda, MD) supplemented with 10% heat-inactivated fetal calf serum (FAKOLA, Bern, Switzerland) and 2 mM L-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 (4–7 M[/omega]) contained 140 mM NaCl, 10 mM HEPES, and 11 mM Fig. 1. 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 mM (P2X2, P2X4, and P2X7). Currents shown in the presence of TNP-ATP are after 4-min application; currents shown after TNPATP are at 4-min wash except for P2X1, in which case the washout was for 8 min. Fig. 2. 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 6 standard error of 4–8 experiments. Lines, least-square fits to a logistic equation (see Experimental Procedures). 970 Virginio et al. at A PE T Jornals on M ay 7, 2017 m oharm .aspeurnals.org D ow nladed from EGTA. The external solution was 147 mM NaCl, 10 mM HEPES, 12 mM glucose, 2 mM KCl, 2 mM CaCl2, and 1 mM MgCl2. 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 260 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 abmeATP were used at the P2X3 receptor and only abmeATP 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.,