Introduction

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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 (P2X_1-7) 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 P2X₃ and heteromeric P2X_2/3 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 P2X_2/3 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 in Xenopus 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 P2X₃ receptor activity. The allosteric actions of cibacron blue also may contribute to increasing the rate of P2X₃ receptor recovery from agonist-induced desensitization.

	Experimental Procedures

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Materials. ATP, 2-methylthio-ATP tetrasodium (2-meSATP), and -methylene ATP dilithium (-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 · ml¹ 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 · ml¹ glucose and 3.6 mg · l¹ 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 rP2X₃ receptor 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 · ml¹ G418; and 1321rX₂-1 cells, 100 µg · ml¹ hygromycin. 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.

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 M) 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

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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 (E_max), 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 hP2X₄ response was observed.

View larger version (12K): [in this window] [in a new window]   Fig. 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, Xenopus oocyte 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.

View larger version (17K): [in this window] [in a new window]   Fig. 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).

View larger version (19K): [in this window] [in a new window]   Fig. 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 -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.

View larger version (19K): [in this window] [in a new window]   Fig. 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-hP2X3 cells: , 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 (E_max = 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 hP2X₃ receptor.

View larger version (20K): [in this window] [in a new window]   Fig. 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).

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).

View larger version (14K): [in this window] [in a new window]   Fig. 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).

View larger version (15K): [in this window] [in a new window]   Fig. 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.

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	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

P2X receptors are members of a group of ligand-gated ion channels, including the -aminobutyric acid_A 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 -aminobutyric acid_A and N-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 P2X₄ receptors.

The mechanism of cibacron blue-mediated P2X₃ receptor 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 hP2X₃ receptor 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 P2X₃ receptor alone.

In addition to ATP, cibacron blue potentiated hP2X₃ receptor activation by several nucleotide analogs, including 2-meSATP, BzATP, and -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 hP2X₃ receptor, 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 rP2X₃ receptor 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 P2X₃ receptors 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 (T_1/2) (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 hP2X₃ receptor resensitization? In the experiments described herein, the concomitant effects of cibacron blue on the E_max of the hP2X₃ receptor 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 (T_1/2 = 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 P2X₃ receptor 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.