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 T_1/2 = 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.