[3H]A-317491, a Novel High-Affinity Non-Nucleotide Antagonist That Specifically Labels Human P2X2/3and P2X3Receptors
- 15 March 2004
- journal article
- Published by American Society for Pharmacology & Experimental Therapeutics (ASPET) in Journal of Pharmacology and Experimental Therapeutics
- Vol. 310 (1), 407-416
- https://doi.org/10.1124/jpet.103.064907
Abstract
A-317491 is a potent and selective antagonist of P2X3 and P2X2/3 receptors. In the present studies, the ability of [3H]A-317491 to label recombinant human P2X2/3 and P2X3 receptors was characterized. Using membranes prepared from 1321N1 cells expressing P2X2/3 receptors, [3H]A-317491 specifically labeled high-affinity (Kd = 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 (Kd = 9 nM) binding sites. Lower affinity binding components (Kd 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 P2X2/3 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 P2X2/3 and P2X3 receptors. The pharmacological profile for P2X receptor antagonists to inhibit [3H]A-317491 binding to P2X2/3 and P2X3 receptors was highly correlated (r = 0.98, P < 0.05), and a similar rank order of potency was observed for blockade of P2X2/3 receptor-mediated calcium influx. These data demonstrate that [3H]A-317491 is the first useful radioligand for the specific labeling of P2X3-containing channels.Keywords
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