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Research ArticleArticle

Allosteric Modulation and Accelerated Resensitization of Human P2X3 Receptors by Cibacron Blue

Karen Alexander, Wende Niforatos, Bruce Bianchi, Edward C. Burgard, Kevin J. Lynch, Elizabeth A. Kowaluk, Michael F. Jarvis and Tim van Biesen
Journal of Pharmacology and Experimental Therapeutics December 1999, 291 (3) 1135-1142;

Abstract

The activity of ATP as a fast neurotransmitter is mediated by the P2X family of ligand-gated ion channels. P2X receptor subtypes are subject to functional modulation by a diverse set of factors, including pH, divalent cations, and temperature. The human P2X3(hP2X3) receptor subunit is expressed primarily in sensory ganglia where it exists as either a homomultimeric receptor or, in combination with P2X2, as a heteromultimeric receptor. This article describes the allosteric modulatory effect of the putative P2X receptor antagonist cibacron blue on the activity of recombinant hP2X3 receptors. In 1321N1 cells expressing the hP2X3 receptor, cibacron blue mediated a 3- to 7-fold increase in both the magnitude and the potency of ATP-activated Ca2+ influx and transmembrane currents. The half-maximal concentration of cibacron blue required to mediate maximal potentiation (EC50 = 1.4 μM) was independent of the agonist used to activate the hP2X3 receptor. The nonselective P2 receptor antagonist PPADS (pyridoxal-5-phosphate-6-azophenyl-2′,4′-disulfonic acid) caused a rightward shift of the cibacron blue concentration-effect curve, whereas increasing concentrations of cibacron blue attenuated PPADS antagonism. In addition to potentiating the effects of ATP at the hP2X3 receptor, cibacron blue also produced a 6-fold increase in the rate of hP2X3receptor recovery from desensitization (from T1/2 = 15.9 to 2.6 min), as evidenced by its ability to restore ATP responsiveness to acutely desensitized receptors. Consistent with the properties of other ligand-gated ion channels, these results suggest that hP2X3 receptor activity can be allosterically modulated by a ligand distinct from the endogenous agonist.

The activity of ATP as a neurotransmitter and intercellular signaling molecule is mediated by a family of ionotropic (P2X) and metabotropic (P2Y) receptors. To date, seven functional P2X receptors (P2X1–7) have been identified by molecular cloning, all of which are characterized by a structural motif consisting of two transmembrane domains joined by an extracellular loop (Ralevic and Burnstock, 1998). P2X receptors function as homomultimeric cation-permeable ion channels and, in some cases, as heteromeric channels consisting of two different P2X receptor subtypes (Lewis et al., 1995; Lê et al., 1998; Torres et al., 1998). At least one pair of P2X receptor subtypes, P2X2 and P2X3, functions as a heteromeric channel in rat nodose ganglion neurons where it exhibits distinct pharmacological and electrophysiological properties (Lewis et al., 1995).

P2X receptor activity is sensitive to modulation by several factors, including pH, divalent cations, and temperature. For example, the pH of the extracellular medium has been found to modulate ATP-mediated signaling in cells expressing the homomeric P2X3and heteromeric P2X2/3 receptors (Stoop et al., 1997). These data corroborate the observation that pH potentiates ATP-mediated membrane currents in rat nodose and dorsal root ganglion neurons (Li et al., 1996a,b), which express endogenous P2X3 and P2X2/3 receptors (Lewis et al., 1995; Burgard et al., 1999).

Recently, extracellular Ca2+ has been shown to accelerate recovery of rat P2X3(rP2X3) receptors from desensitization (Cook et al., 1998). Elevated levels of extracellular multivalent cations, including Ca2+, Ba2+, and Gd3+ reportedly lead to a larger available pool of agonist-sensitive receptors by increasing the rate of recovery from desensitization, thereby increasing the apparent magnitude of the transmembrane currents (Cook et al., 1998). They postulate that Ca2+ binding to the extracellular surface of the receptor plays a role in short-term modulatory mechanisms that permit delayed responses to transient signals.

The utility of cibacron blue, an anthraquinone sulfonic acid derivative, as an inhibitor of ATP-mediated signaling and of P2X and P2Y receptor activation has been well documented (Ralevic and Burnstock, 1998). Cibacron blue functions as an antagonist of several diverse ATP-mediated physiological responses, including rat urinary bladder smooth muscle contraction (Hashimoto and Kokubun, 1995), rat cecum inhibitory junction potentials (Manzini et al., 1986), phospholipid secretion from rat isolated alveolar type II cells (Rice and Singleton, 1989), and calcium influx in rat parotid acinar cells (Soltoff et al., 1989). Cibacron blue also functions both as an antagonist of P2 receptor-operated inward currents and calcium influx in PC12 cells (Nakazawa et al., 1991; Michel et al., 1996; Surprenant, 1996), and as an inhibitor of ecto-nucleotidase activity inXenopus oocytes (Ziganshin et al., 1996). Recombinant rP2X1 and P2X2 receptors also are sensitive to inhibition by cibacron blue (Surprenant, 1996). In vivo, cibacron blue functions as an inhibitor of ATP-induced inflammation of the mouse hind paw (Ziganshina et al., 1996).

Although the effects of cibacron blue appear to be primarily inhibitory, one study has described its potentiating activity at the P2X4 receptor (Miller et al., 1998). In human embryonic kidney cells (HEK) 293 cells expressing the rP2X4 receptor, pretreatment with cibacron blue mediated a 4-fold increase in the potency of ATP without affecting the maximum response (Miller et al., 1998).

This article describes the potentiation of homomultimeric human P2X3 (hP2X3) receptor-mediated Ca2+ influx and transmembrane currents by cibacron blue. The effects of cibacron blue on both the efficacy and potency of P2X3 receptor agonists and antagonists are described. Electrophysiological and Ca2+ influx data suggest that cibacron blue functions as an allosteric modulator of P2X3receptor activity. The allosteric actions of cibacron blue also may contribute to increasing the rate of P2X3receptor recovery from agonist-induced desensitization.

Experimental Procedures

Materials.

ATP, 2-methylthio-ATP tetrasodium (2-meSATP), and αβ-methylene ATP dilithium (αβ-meATP) were obtained from Research Biochemicals Inc. (Natick, MA). 2′- and 3′-O-(4-Benzoylbenzoyl)-ATP tetraethylammonium salt (mixed isomers) (BzATP) was obtained from Sigma Chemical Co. (St. Louis, MO). G418 sulfate was obtained from Calbiochem-Novabiochem Corp. (La Jolla, CA). Dulbecco's modified Eagle's medium (with 4.5 mg · ml−1 glucose and 4 mM l-glutamine) and fetal bovine serum were obtained from Hy-Clone Laboratories Inc. (Logan, UT). Dulbecco's PBS (with 1 mg · ml−1 glucose and 3.6 mg · l−1 Na pyruvate, without phenol red), hygromycin, and lipofectamine were obtained from Life Technologies, Inc. (Grand Island, NY). Fluo-4 AM was purchased from Molecular Probes, Inc. (Eugene, OR).

Stable Cell Lines and Cell Culture.

The rP2X3receptor cDNA was 100% identical with the previously published sequence (Garcia-Guzman et al., 1997). The hP2X3 receptor was essentially identical with that reported by Garcia-Guzman et al. (1997) (GenBank accession no. Y07683). A single exception was at amino acid residue 126, where an arginine was encoded; the published sequence encodes a proline at this position. Multiple replications of cloning the hP2X3 receptor yielded the same sequence, suggesting that the observed difference is not the result of a cloning artifact or a sequencing error. The 1321N1 human astrocytoma cells stably expressing rP2X3 or hP2X3 receptors (1321rX3-3 and 1321hX3-11, respectively) were constructed with standard lipid-mediated transfection methods. All cell lines were maintained in Dulbecco's PBS containing 10% fetal bovine serum and antibiotics as follows: 1321rX3-3 and 1321hX3-11 cells, 300 μg · ml−1 G418; and 1321rX2-1 cells, 100 μg · ml−1hygromycin. Cells were grown at 37°C in a humidified atmosphere containing 5% CO2.

Measurement of Intracellular Ca2+ Levels.

P2X receptor function was determined on the basis of agonist-mediated increases in cytosolic Ca2+ concentration. Briefly, a fluorescent Ca2+ chelating dye (fluo-4) was used as an indicator of the relative levels of intracellular Ca2+ in a 96-well format with a fluorescence imaging plate reader (Molecular Devices Corp., Menlo Park, CA). Cells were grown to confluence in 96-well black-walled tissue culture plates and loaded with the acetoxymethylester form of fluo-4 (1 μM) in Dulbecco's PBS for 1–2 h at 23°C. Cibacron blue (50 μl of 4× concentration) was added 3 min before the addition of agonists (50 μl of 4× concentration) (final volume 200 μl). Fluorescence data were collected at 1- to 5-s intervals throughout each experimental run.

Data shown are based on the peak increase in relative fluorescence units compared with basal fluorescence. Concentration-effect curves for all cell types are shown as a percentage of the maximum ATP-mediated signal measured in the absence of cibacron blue. Concentration response data were analyzed with a four-parameter logistic Hill equation in GraphPad Prism (San Diego, CA). All data are expressed as means ± S.E. Statistical analysis was performed with Student's ttest (P < .05) on the basis of pIC50 values.

Electrophysiology.

The hP2X3 receptor subtype expressed in Xenopus oocytes was characterized with standard two-electrode voltage-clamp techniques. Briefly, oocytes were denuded of overlying follicle cells and intranuclear injections of 12 nl of cDNA (1 μg/μl) were performed on each oocyte. Oocytes were used for recordings 1 to 5 days postinjection and were perfused (3.5 ml/min) with a standard recording solution containing 96 mM NaCl, 2.0 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, 5.0 mM Na-pyruvate, and 5.0 mM Na-HEPES (pH 7.4). Electrodes (1.5–2.0 MΩ) were filled with 120 mM KCl. ATP was applied with a solenoid-driven drug application pipette positioned close to the oocyte in the perfusion chamber. ATP was applied every 3.5 min, and application duration typically lasted 5 s. Cibacron blue was bath-applied for at least 3 min before being coapplied with ATP through the drug pipette. Cells were voltage-clamped at −60 mV. Data were acquired and analyzed with pClamp software (Axon Instruments, Inc, Foster City, CA).

Results

Cibacron Blue Potentiates ATP-Activated hP2X3 Receptor Responses.

hP2X3 receptor-mediated responses were determined in stably transfected 1321N1 cells by measuring the magnitude of ATP-activated Ca2+ flux into the cytosol (Fig.1A). ATP activation caused a rapid and transient increase in the levels of cytoplasmic Ca2+. The shapes of the Ca2+ influx curves were qualitatively similar to electrophysiological data measured in Xenopus oocytes (Fig. 1B) and were consistent with previously reported observations (Bianchi et al., 1999). Preincubation of the cells for 3 min with cibacron blue (10 μM) led to a 3- to 7-fold increase in the magnitude of the maximal ATP-activated response (Emax), as measured both by Ca2+influx (Fig. 1A) and transmembrane currents (Fig. 1B). Cibacron blue mediated a similar 3- to 7-fold potentiation of the maximal ATP response with cells expressing the rP2X3 receptor homolog (data not shown). Pilot experiments showed that the onset of the cibacron blue effect occurred in <1 min, thus a 3-min preapplication time was selected to ensure full activity. Cibacron blue alone exhibited no intrinsic effect on Ca2+ influx at concentrations up to 200 μM and did not measurably affect the pH of the assay buffer (pH 7.2) at concentrations up to 1 mM. The potentiating effect of cibacron blue was specific for the P2X3 receptor because concentrations of cibacron blue up to 1 mM did not alter agonist activation of hP2X1, hP2X2, and hP2X7 receptors expressed in 1321N1 cells (data not shown). Cibacron blue (10 μM) did enhance the potency of ATP activation of hP2X4 receptor-mediated Ca2+ influx in the presence of submaximal concentrations of agonist, as has previously been described (Miller et al., 1998). However, no increase in the maximal ATP-activated hP2X4response was observed.

Figure 1
Figure 1

Calcium influx mediated by ATP-stimulated hP2X3 receptors is potentiated by cibacron blue. A, 1321-P2X3 cells loaded with the Ca2+ indicator fluo-4 were treated with 3 μM ATP (t = 210 s) in the presence (solid line) or absence (dashed line) of 10 μM cibacron blue (added at t = 10 s). Relative fluorescence is shown as the percentage of the maximum response obtained in the absence of cibacron blue. B, Xenopusoocyte expressing hP2X3 receptors was challenged with 1 μM ATP in the absence (small current) and presence (large current) of cibacron blue (1 μM). ATP application is denoted by the horizontal bar. ATP was applied 3.5 min apart, with cibacron blue present during the interapplication interval and then coapplied with ATP.Vh = −60 mV.

In electrophysiological studies (n = 6), cibacron blue (1 μM) produced a potentiation of the peak amplitude of 1 μM ATP-activated currents to 213 ± 49% of control (Fig. 1B). The effect of cibacron blue on the Emax of the hP2X3 receptor-mediated transmembrane current was long-lasting, such that full potentiation was observed up to 9 min after a brief (1 min) exposure to cibacron blue. The onset of the cibacron blue potentiation effect was rapid (<1 min; data not shown). Coapplication of cibacron blue and ATP resulted in potentiation of the Ca2+ influx signal, albeit with lower apparent potency and Emax than observed after a 3-min preincubation period. Cibacron blue (10 μM) had no apparent effect on the kinetics of the ATP-activated Ca2+flux response (Fig. 1A) or on the acute desensitization kinetics of the hP2X3 receptor (Fig. 1B).

The potentiation of ATP-activated hP2X3 receptors by cibacron blue was concentration-dependent (Fig.2), with an observed half-maximal response (EC50) of 1.4 ± 0.5 μM (Fig.3). In addition to increasing theEmax of the ATP-activated hP2X3 receptor response, cibacron blue also caused a concentration-dependent leftward shift of the ATP concentration-effect curve (Fig. 2). In the presence of 3 μM cibacron blue, the magnitude of ATP-activated hP2X3receptor signaling was increased >3-fold (Emax = 330 ± 5%), whereas the EC50 of ATP was decreased from 356 ± 100 nM to 46 ± 8 nM (Fig. 2).

Figure 2
Figure 2

Cibacron blue (CB) significantly increases the potency of ATP-induced hP2X3 receptor activation in a concentration-dependent manner. ATP concentration-effect curves were determined in the absence or presence of cibacron blue by measuring Ca2+ influx, as determined by fluo-4 fluorescence, in 1321N1-hP2X3 cells: ▪, without cibacron blue (ATP EC50 = 356 ± 47 nM;Emax = 102 ± 3%); ▴, 1 μM cibacron blue (ATP EC50 = 64 ± 7 nM*; Emax = 267 ± 6%*); ▾, 3 μM cibacron blue (ATP EC50= 46 ± 8 nM*; Emax = 330 ± 5%*); ♦, 10 μM cibacron blue (ATP EC50 = 60 ± 12 nM*;Emax = 345 ± 6%*). Data are shown as a percentage of the maximal response to 10 μM ATP and are the means ± S.E. of three experiments (statistical analysis based on pEC50 values; *P < .05 compared with control).

Figure 3
Figure 3

The potency of cibacron blue to potentiate hP2X3 receptor activation is similar for prototypic P2X3 agonists. Cibacron blue concentration-effect curves were determined for each of four prototypic P2X3 receptor agonists by measuring Ca2+ influx, as determined by fluo-4 fluorescence, in 1321N1-hP2X3 cells. The half-maximal concentrations of cibacron blue required to mediate full potentiation were as follows: ▾, 10 μM ATP (cibacron blue EC50= 1.4 ± 0.5 μM; Emax = 504 ± 15%); ▴, 10 μM 2-meSATP (cibacron blue EC50 = 1.4 ± 0.2 μM; Emax = 555 ± 18%); ▪, 10 μM BzATP (cibacron blue EC50 = 0.9 ± 0.1 μM; Emax = 562 ± 12%); ♦, 10 μM αβ-meATP (cibacron blue EC50 = 1.4 ± 0.2 μM;Emax = 537 ± 14%). Data are shown as a percentage of the maximal response to 10 μM ATP and are the means ± S.E. of three experiments. Concentration-effect curves were fitted with a four-parameter logistic equation in GraphPad Prism.

The EC50 of cibacron blue required to mediate potentiation was similar irrespective of the agonist used to activate hP2X3 receptors. TheEmax of hP2X3receptor activation by maximal (10 μM) concentrations of either ATP, BzATP, 2-meSATP, or αβ-meATP, all of which are known agonists for the P2X3 receptor, was similar at all cibacron blue concentrations (Fig. 3). Cibacron blue did not confer agonist activity to nucleotides previously shown to be inactive at the P2X3 receptor (Garcia-Guzman et al., 1997;Bianchi et al., 1999), including ADP, UTP, and UDP (100 μM; data not shown). The potentiating effect of cibacron blue was unaffected by depletion of intracellular Ca2+ stores with thapsigargin but was completely abrogated in the presence of excess extracellular ethylene glycol bis(β-aininoethyl ether)-N,N,N′,N′,-tetraacetic acid, suggesting that the increased magnitude of the ATP-activated response was due to increased Ca2+ flow across the plasma membrane (data not shown).

Triazene dyes structurally related to cibacron blue, including basilen blue, reactive blue 5, reactive red 2, reactive orange 14, and reactive yellow 2 were tested for their ability to potentiate hP2X3 receptor activation by ATP (Fig.4). Whereas reactive orange 14 and reactive yellow 2 exhibited little or no potentiating activity, basilen blue, reactive blue 5, and reactive red 2 mediated significant hP2X3 receptor potentiation. The anthraquinone sulfonic acid derivatives basilen blue and reactive blue 5 exhibited half-maximal concentrations of hP2X3 receptor potentiation similar to cibacron blue (EC50 = 1.2 ± 0.6 μM and 1.4 ± 0.5 μM, respectively). Reactive red 2 was significantly less potent as a potentiator of hP2X3 receptor activation by ATP (EC50 = 55 ± 10 μM) (Fig. 4). None of the triazene dyes tested were intrinsically fluorescent, nor did they affect the pH of the assay buffer at concentrations up to 1 mM.

Figure 4
Figure 4

Potentiation of hP2X3 receptor activity with various triazene dyes. Concentration-effect curves for four structurally related triazene dyes were determined by measuring ATP-activated Ca2+ influx in 1321N1-hP2X3cells: ▪, reactive red 2 (EC50 = 55 ± 10 μM;Emax = 600%, fixed parameter); ♦, basilen blue (EC50 = 1.2 ± 0.6 μM;Emax = 373 ± 17%*); ▴, reactive blue 5 (EC50 = 1.4 ± 0.5 μM;Emax = 534 ± 14%); ▾, cibacron blue (EC50 = 1.2 ± 0.2 μM;Emax = 566 ± 17%). Data are shown as a percentage of the maximal response to 10 μM ATP and are the means ± S.E. of three experiments (statistical analysis based on pEC50 values; *P < .05 compared with control).

Cibacron Blue Blocks Inhibitory Activity of PPADS.

The inhibition of hP2X3 receptors by PPADS (pyridoxal-5-phosphate-6-azophenyl-2′,4′-disulfonic acid), a nonselective P2 receptor antagonist, has been demonstrated previously (Garcia-Guzman et al., 1997). In the absence of cibacron blue, PPADS inhibited ATP-mediated hP2X3 activation with a half-maximal concentration (IC50) of 8.6 ± 3 μM (Fig.5A). Pretreatment of the hP2X3-expressing cells with 10 μM cibacron blue increased both the maximal ATP-activated signal (Emax = 437 ± 6%) and the apparent IC50 (51 ± 3 μM) of PPADS. To determine whether cibacron blue mediates this effect by increasing the effective potency of ATP, the experiment was performed with 1, 3, 10, or 30 μM ATP. For all concentrations of ATP, cibacron blue produced a similar concentration-dependent rightward shift of the PPADS concentration-effect curves. For example, in the absence of cibacron blue, the apparent IC50 values for PPADS at each ATP concentration were 3.64 ± 1.1 μM (1 μM ATP), 3.11 ± 1.0 μM (3 μM ATP), 4.81 ± 1.1 μM (10 μM ATP), and 2.67 ± 0.7 μM (30 μM ATP) respectively, confirming that PPADS is a noncompetitive antagonist at the P2X3 receptor. Similarly, PPADS was found to be noncompetitive with ATP at concentrations of cibacron blue up to 100 μM (data not shown). The effect of cibacron blue on the inhibitory potency of PPADS was thus found to be independent of ATP concentration, suggesting that cibacron blue and PPADS exhibit mutually exclusive effects at the hP2X3receptor.

Figure 5
Figure 5

Cibacron blue blocks the inhibitory activity of PPADS. A, concentration-effect curves for the inhibition of ATP-activated hP2X3 receptor activity by PPADS were determined in the presence and absence of cibacron blue. PPADS and cibacron blue were coapplied 3 min before the addition of 3 μM ATP: ▪, without cibacron blue (PPADS IC50 = 8.6 ± 3 μM; Emax = 101 ± 4%); ●, 1 μM cibacron blue (PPADS IC50 = 14 ± 3 μM;Emax = 280 ± 6%*); ♦, 10 μM cibacron blue (PPADS IC50 = 51 ± 4 μM*; Emax = 437 ± 6%*); ▴, 100 μM cibacron blue (PPADS IC50 = 220 ± 186 μM*;Emax = 488 ± 9%*). Inset, data are normalized to the maximal signal observed at each concentration of cibacron blue. B, concentration-effect curves for cibacron blue potentiation of hP2X3 responses, activated by 3 μM ATP, were determined in the presence and absence of PPADS: ▪, without PPADS (cibacron blue EC50 = 3.8 ± 0.4 μM; Emax = 738 ± 22%); ▴, 5 μM PPADS (cibacron blue EC50 = 4.5 ± 0.3 μM,Emax = 682 ± 15%); ▾, 10 μM PPADS (cibacron blue EC50 = 7.5 ± 0.2 μM*; Emax = 730 ± 7%);♦, 50 μM PPADS (cibacron blue EC50 = 15 ± 1.4 μM*; Emax = 653 ± 10%). Data are shown as a percentage of the maximal response to 3 μM ATP and are the means ± S.E. of three experiments (statistical analysis based on pEC50 values; *P < .05 compared with control).

In the converse of this experiment, the effect of PPADS on cibacron blue potentiation of the hP2X3 receptor was determined (Fig. 5B). PPADS caused a concentration-dependent rightward shift of the concentration-effect curve of cibacron blue, while simultaneously reducing the initial magnitude of ATP activation (Fig.5B). Although 50 μM PPADS was sufficient to fully inhibit ATP-activated hP2X3 receptors, cibacron blue overcame the inhibitory activity of PPADS in a concentration-dependent manner.

Cibacron Blue Accelerates Rate of hP2X3 Receptor Resensitization.

The potency of cibacron blue as a modulator of hP2X3 receptor activity was determined in nondesensitized and acutely desensitized receptors (Fig.6). The 1321N1-hP2X3 cells were exposed to 10 μM ATP for 1 min to acutely desensitize the hP2X3 receptors. As described in Fig. 2, the EC50 of cibacron blue required to fully potentiate nondesensitized hP2X3 receptors was 1.1 ± 0.2 μM (Fig. 6). However, acutely desensitized hP2X3 receptors appeared to be less sensitive to cibacron blue-mediated potentiation (EC50 = 6.4 ± 0.5 μM), such that 100 μM cibacron blue was required to achieve a maximal signal. Regardless of the initial state of the hP2X3 receptors (nondesensitized or acutely desensitized), cibacron blue pretreatment ultimately led to a similar agonist-activated maximal activity, suggesting that the size of the receptor pool was comparable under both conditions (Fig. 6).

Figure 6
Figure 6

Cibacron blue significantly increases the rate of hP2X3 receptor recovery from desensitization. Cibacron blue concentration-effect curves were determined in nondesensitized and acutely desensitized 1321-hP2X3 cells: ▪, nondesensitized (cibacron blue EC50 = 1.1 ± 0.1 μM;Emax = 288 ± 5%); ●, desensitized (cibacron blue EC50 = 6.4 ± 0.4 μM*; Emax = 302 ± 5%). Data are shown as a percentage of the maximal response to 3 μM ATP and are the means ± S.E. of three experiments (statistical analysis based on pEC50 values; *P < .05 compared with control).

The activity of cibacron blue to restore functional activation to acutely desensitized receptors was investigated. The hP2X3 receptor-expressing cells were desensitized by pretreatment with ATP (10 μM) for 1 min, washed to remove extracellular ATP, and after various time periods of incubation with or without cibacron blue, desensitized receptors were rechallenged with ATP. Figure 7a demonstrates the lack of hP2X3 response to a second challenge with ATP immediately after desensitization (1.5 min). Extension of the incubation time between desensitization and subsequent challenge with ATP revealed the progressive recovery of hP2X3receptor activity, approaching the control (nondesensitized) signal by 61.5 min.

Figure 7
Figure 7

Cibacron blue accelerates the recovery of hP2X3 receptors from acute desensitization. A, 1321-hP2X3 cells were pretreated with 10 μM ATP or Dulbecco's PBS (control curve) for 1 min, washed twice to remove excess extracellular ATP, and incubated for the time periods shown before rechallenge with various concentrations of ATP. The control curve (dashed line) shows the concentration-effect of ATP on mock-desensitized (Dulbecco's PBS-treated) cells. B, 1321-hP2X3 cells were pretreated with 10 μM ATP for 1 min, washed twice to remove excess extracellular ATP, and incubated for the time periods shown in the presence of 50 μM cibacron blue before rechallenge with various concentrations of ATP. The control curve (dashed line) shows the concentration-effect of ATP on mock-desensitized (Dulbecco's PBS-treated) cells pretreated with 50 μM cibacron blue. C, rates of receptor recovery are shown as a function of percentage of the nondesentized response over time. Curves are solutions of %control = max(1 − exp(−Kt*time)), where %control is the percentage of receptor activity compared with nondesensitized receptors, max is the %control activity observed at 61.5 min, time is the time in minutes, and Kt is the time constant. T1/2 (half time of receptor resensitization) was calculated as ln(0.5)/−K.

The addition of 50 μM cibacron blue during the incubation period following ATP-induced desensitization appeared to increase both the apparent potency of ATP and the rate of recovery from desensitization (Fig. 7B). After a 15-min incubation with cibacron blue, the desensitized cells showed almost full activity compared with control (nondesensitized) cells, indicating a considerably shorter refractory period after desensitization. Note that the inclusion of cibacron blue in the incubation buffer leads to enhanced rate of recovery of hP2X3 receptors from desensitization, increased final Emax, and increased potency of the agonist (Fig. 7B).

Figure 7C shows the maximal receptor signal at various time points following acute desensitization as a percentage of the control (nondesensitized) signal in the presence and absence of 50 μM cibacron blue (see dashed lines in Fig. 7, A and B). The calculated half times (T1/2) of the refractory period (defined as the time required to restore 50% of the activity observed at 60 min) were 15.9 min (Kt = 0.0436 min−1) in the absence and 2.6 min (Kt = 0.2626 min−1) in the presence of cibacron blue. Thus, cibacron blue increases the rate of hP2X3 receptor recovery from desensitization by 6-fold.

Discussion

P2X receptors are members of a group of ligand-gated ion channels, including the γ-aminobutyric acidA receptor, the N-methyl-d-aspartate receptor, the 5-hydroxytryptamine3 receptor, and the nicotinic acetylcholine receptors, which play a role in neurotransmission. Many of these receptors have been shown to be the targets of allosteric modulatory mechanisms, including γ-aminobutyric acidA andN-methyl-d-aspartate receptors (Robichon et al., 1997; Sigel and Buhr, 1997). Allosteric modulators of receptor activity generally enhance agonist-induced receptor activation by binding to secondary sites on the receptor.

This article describes the effect of cibacron blue on both the magnitude of the hP2X3 receptor response and the potency of hP2X3 receptor agonists and antagonists. In 1321N1 cells stably transfected with the hP2X3 receptor, exposure to cibacron blue led to a 3- to 7-fold increase in the magnitude of the maximal ATP-activated Ca2+ influx. Similar data were observed with the rat homolog of the P2X3 receptor, suggesting that the modulatory activity of cibacron blue is not species dependent. The potentiating effect of cibacron blue might be due to either 1) the increased ability of P2X3 receptors to conduct ions by increasing conductance, channel open time, or channel opening frequency; 2) the activation of a larger number of P2X3 receptors per cell; or 3) enhancement of the cooperativity of agonist binding and receptor activation [cooperativity of ATP binding has previously been shown for P2X2 receptor activation (Ding and Sachs, 1999)].

An allosteric modulatory mechanism has been proposed for cibacron blue at the rP2X4 receptor (Miller et al., 1998). Cibacron blue was previously shown to increase the apparent potency of ATP-mediated activation of the rP2X4 receptor by 4-fold. The potency of cibacron blue-mediated rP2X4 receptor potentiation (EC50 = 3.3 μM) was comparable to that described herein for the hP2X3 receptor. However, the mechanism of potentiation differs with respect to the effect of cibacron blue on the maximal signals. Unlike its effects at the hP2X3 receptor, cibacron blue did not promote supermaximal activation of rP2X4 receptors in the presence of saturating agonist concentrations (Miller et al., 1998). Also, simultaneous application of cibacron blue with agonist resulted in the inhibition of rP2X4 receptor activation, whereas hP2X3 receptor activity is potentiated under these conditions. These discrepant observations suggest a mechanistic difference between the modulatory activities of cibacron blue at the P2X3 and P2X4receptors.

The mechanism of cibacron blue-mediated P2X3receptor potentiation is not a result of its previously described inhibitory effect on ectonucleotidases (IC50 = 44 μM) (Stout and Kirley, 1995). If cibacron blue-mediated ecto-ATPase activity were a contributing factor, it would be expected that cibacron blue alone would mediate agonist-like activity by increasing the level of endogenous ATP in the medium. However, the present data reveal no intrinsic effects of cibacron blue on hP2X3receptor activity. Furthermore, the accumulation of endogenous agonist as a result of ecto-ATPase inhibition would be expected to affect all P2 receptor subtypes, rather than the P2X3receptor alone.

In addition to ATP, cibacron blue potentiated hP2X3 receptor activation by several nucleotide analogs, including 2-meSATP, BzATP, and αβ-meATP. In each case, the half-maximal concentration of cibacron blue required to mediate full potentiation was similar, suggesting that the effect of cibacron blue on the receptor was independent of the agonist. In addition to mediating an increase in the magnitude of the maximal P2X3 receptor signal, cibacron blue caused a leftward shift of the ATP concentration-response curve. In the presence of 3 μM cibacron blue, ATP was 7-fold more potent than in its absence, suggesting that cibacron blue may have an affect on the affinity and/or the efficacy of ATP for the hP2X3receptor, or serves to enhance the cooperativity of ATP binding to the multimeric receptor. Although the exact stoichiometric organization of the multimeric P2X3 receptor remains unknown, recent reports provide structural and pharmacological evidence that P2X receptors may exist as trimers (Nicke et al., 1998; Ding and Sachs, 1999) or tetramers (Kim et al., 1997) that exhibit positive ATP-binding cooperativity.

The modulatory activity of cibacron blue was corroborated by the observation that the inhibitory potency of a noncompetitive P2X3 antagonist, PPADS, was inversely related to the concentration of cibacron blue. Cibacron blue, although increasing the magnitude of P2X3 receptor activation, caused a rightward shift of the PPADS concentration-effect curve. This effect of cibacron blue was independent of ATP concentration and is thus not a consequence of an apparent increase in receptor occupancy. The cibacron blue-mediated leftward shift of the agonist concentration-effect curves and rightward shift of the antagonist concentration-effect curves support the conclusion that cibacron blue functions as an allosteric modulator of P2X3 receptor activity. Furthermore, the mutual exclusivity of PPADS-mediated inhibition and cibacron blue-mediated potentiation suggests a complex interaction between regulatory ligands that modulate P2X3 receptor function.

The observation that the modulatory effects of cibacron blue at the hP2X3 receptor are long-lasting is similar to that described for the modulatory effects of Ca2+at the rP2X3 receptor (Cook et al., 1998) and cibacron blue at the rP2X4 receptor (Miller et al., 1998). This may be due to either slow reversibility of cibacron blue binding to its putative binding site on the receptor, or to long-lasting changes of the conformational state or desensitization state of the receptor.

The potentiating effect of cibacron blue at the P2X3 receptor is not likely to be due an increase in the absolute number of receptors on the cell surface because its onset of action is rapid (<1 min) and thus precludes the de novo synthesis of receptor protein. Similarly, desensitization of the hP2X3 receptors does not appear to lead to a change in the receptor density, as evidenced by the ability of cibacron blue to fully potentiate the hP2X3 signal to maximal levels in both desensitized and nondesensitized cells.

Previously, Cook et al. (1998) described the effect of Ca2+ on the rate of rP2X3receptor recovery from desensitization. Pretreatment with high concentrations of Ca2+ (1–10 mM) were shown to accelerate receptor resensitization by 2-fold, via an integrative and long-lasting mechanism. Interestingly, this effect was reported to be considerably weaker at the hP2X3 receptor, an observation that we have confirmed (data not shown). However, the modulatory effect of cibacron blue described herein was observed at both rat and hP2X3 receptors, and is at least 1000-fold more potent than the actions of Ca2+, suggesting that endogenously expressed P2X3receptors might be subject to functional regulation by a multiplicity of low- and high-affinity interactions.

The present data indicate that the potentiation of both the human and rP2X3 receptors by cibacron blue occurs concomitantly with accelerated receptor resensitization. The apparent rate of recovery from desensitization is increased 6-fold in the presence of 50 μM cibacron blue. The decrease in the half-life of the refractory period after desensitization (T1/2) (from 15.9 to 2.6 min) suggests that endogenously expressed P2X3 receptors may be subject to modulatory mechanisms that facilitate their functional recovery. This observation is similar to the 2-fold decrease of the refractory period of rP2X3 receptors exposed to high levels of extracellular Ca2+ (Cook et al., 1998).

How then does the allosteric modulatory effect of cibacron blue relate to its ability to accelerate the rate of hP2X3receptor resensitization? In the experiments described herein, the concomitant effects of cibacron blue on theEmax of the hP2X3receptor response and the potency of the agonists were inseparable. However, whereas the potencies of agonists and antagonists were maximally shifted after relatively brief exposures to cibacron blue (<1 min), full recovery from desensitization by cibacron blue requires >10 min (T1/2 = 2.6 min). This temporal separation of the two distinguishable effects of cibacron blue, namely, allosteric modulation and recovery from desensitization, suggests that they may be functionally independent. Although both elevated Ca2+ and cibacron blue appear to engender a comparable effect on receptor resensitization, Ca2+ has not been shown to serve as an allosteric modulator of P2X3 receptors. We postulate that the binding of cibacron blue to the P2X3 receptor leads to a rapid conformational change, resulting in the potentiation of ATP-mediated P2X3 receptor activation. This conformational change also mediates a slower, long-term effect on the desensitization state of the receptor, such that allosteric modulation and functional resensitization occur sequentially and may share a common mechanism of action.

Given that little is known about the desensitization mechanism of P2X3 receptors, it is difficult to differentiate between several possible mechanisms of action for cibacron blue-mediated acceleration of receptor recovery from desensitization, including 1) inhibition of agonist-induced receptor internalization, 2) alteration of the receptor phosphorylation/modification state after agonist activation, and 3) simple acceleration of agonist dissociation from the receptor after activation.

The effects of cibacron blue at the P2X3 receptor provide novel insights into the physical and pharmacological properties of the P2X3 receptor. Although no naturally occuring ligands have been shown to exhibit the modulatory activity of cibacron blue, the observation that the P2X3receptor is subject to regulatory mechanisms that affect both ligand potencies and the desensitization state of the receptor suggests that the regulation of its physiological role as a neurotransmitter receptor may be complex, and could potentially involve a multiplicity of regulatory ligands.

Footnotes

  • Send reprint requests to: Tim van Biesen, Ph.D., Neurological and Urological Diseases Research, D-4PM, AP9A, Abbott Laboratories, Abbott Park, IL 60064-3500. E-mail:tim.vanbiesen{at}abbott.com

  • Abbreviations:
    2-meSATP
    2-methylthio-ATP tetrasodium
    αβ-meATP
    αβ-methylene ATP dilithium
    BzATP
    2′- and 3′-O-(4-benzoylbenzoyl)-ATP tetraethylammonium salt
    PPADS
    pyridoxal-5-phosphate-6-azophenyl-2′,4′-disulfonic acid
    • Received April 27, 1999.
    • Accepted August 17, 1999.

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Allosteric Modulation and Accelerated Resensitization of Human P2X3 Receptors by Cibacron Blue

Karen Alexander, Wende Niforatos, Bruce Bianchi, Edward C. Burgard, Kevin J. Lynch, Elizabeth A. Kowaluk, Michael F. Jarvis and Tim van Biesen
Journal of Pharmacology and Experimental Therapeutics December 1, 1999, 291 (3) 1135-1142;
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