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Rapid desensitization of ligand-gated ion channel receptors can alter the apparent activity of receptor modulators, as well as make detection of fast-channel activation difficult. Investigation of the antagonist pharmacology of ATP-sensitive homomeric P2X3 receptors is limited by agonist-evoked fast-desensitization kinetics. In the present studies, chimeric receptors were created using the coding sequence for the N-terminus and the first transmembrane domain of either the nondesensitizing human P2X2a or fast-desensitizing P2X3 receptor joined to the sequence encoding the extracellular loop, second transmembrane domain, and C-terminus of the other receptor (designated P2X2–3 and P2X3–2, respectively). These clones were stably transfected into 1321N1 astrocytoma cells for biophysical and pharmacological experiments using both electrophysiological and calcium-imaging methods. Chimeric P2X2–3 and P2X3–2 receptors were inwardly rectifying and agonist responses showed desensitization properties similar to the wild-type human P2X2a and P2X3 receptors, respectively. The P2X2–3 chimera displayed an agonist pharmacological profile similar to the P2X3 wild-type receptor being activated by low concentrations of both ATP and α,β-meATP. In contrast, the P2X3–2 chimera had markedly reduced sensitivity to both agonists. The P2X3 receptor antagonists TNP-ATP and A-317491 were shown to be potent, competitive antagonists of the P2X2–3 chimera (Ki=2.2 and 52.1 nM, respectively), supporting the hypothesis that rapid receptor desensitization can mask the competitive antagonism of wild-type homomeric P2X3 receptors. British Journal of Pharmacology (2003) 140, 202–210. doi:10.1038/sj.bjp.0705411