Abstract

A-317491 is a potent and selective antagonist of P2X₃ and P2X_2/3 receptors. In the present studies, the ability of [³H]A-317491 to label recombinant human P2X_2/3 and P2X₃ receptors was characterized. Using membranes prepared from 1321N1 cells expressing P2X_2/3 receptors, [³H]A-317491 specifically labeled high-affinity (K_d = 0.9 nM) recognition sites. High-affinity [³H]A-317491 binding was not detected in membrane preparations from native 1321N1 cells or cells expressing homomeric P2X₁, P2X₂, or P2X₃ receptors. Specific [³H]A-317491 P2X₃ receptors could only be reliably detected following treatment of intact P2X₃ receptor-expressing cells with apyrase (1 U/ml) both before and during membrane preparation. Under these conditions, [³H]A-317491 also labeled high-affinity (K_d = 9 nM) binding sites. Lower affinity binding components (K_d values of 87–790 nM) were detected in both assays using higher ligand concentrations that likely represent nonfunctional recognition sites. [³H]A-317491 binding to both P2X_2/3 and P2X₃ receptors was reversible, and ligand kinetic studies provided similar estimates of the high-affinity binding constants. Potent P2X₃ receptor agonists 2-methylthio-ATP, 2,3-O-(4-benzoylbenzoyl)-ATP, and α,β-methylene adenosine triphosphate also potently inhibited specific [³H]A-317491 binding to both P2X_2/3 and P2X₃ receptors. The pharmacological profile for P2X receptor antagonists to inhibit [³H]A-317491 binding to P2X_2/3 and P2X₃ receptors was highly correlated (r = 0.98, P < 0.05), and a similar rank order of potency was observed for blockade of P2X_2/3 receptor-mediated calcium influx. These data demonstrate that [³H]A-317491 is the first useful radioligand for the specific labeling of P2X₃-containing channels.