Vol. 291, Issue 3, 1135-1142, December 1999

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

Neurological and Urological Diseases Research, Abbott Laboratories, Abbott Park, Illinois

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 hP2X₃ receptor 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.