Competitive Antagonism of Recombinant P2X_2/3Receptors by 2′,3′-O-(2,4,6-Trinitrophenyl) Adenosine 5′-Triphosphate (TNP-ATP)

Abstract

TNP-ATP has become widely recognized as a potent and selective P2X receptor antagonist, and is currently being used to discriminate between subtypes of P2X receptors in a variety of tissues. We have investigated the ability of TNP-ATP to inhibit α,β-methylene ATP (α,β-meATP)-evoked responses in 1321N1 human astrocytoma cells expressing recombinant rat or human P2X_2/3 receptors. Pharmacological responses were measured using electrophysiological and calcium imaging techniques. TNP-ATP was a potent inhibitor of P2X_2/3 receptors, blocking both rat and human receptors with IC₅₀ values of 3 to 6 nM. In competition studies, 10 to 1000 μM α,β-meATP was able to overcome TNP-ATP inhibition. Schild analysis revealed that TNP-ATP was a competitive antagonist with pA₂ values of −8.7 and −8.2. Inhibition of P2X_2/3 receptors by TNP-ATP was rapid in onset, reversible, and did not display use dependence. Although the onset kinetics of inhibition were concentration-dependent, the TNP-ATP off-kinetics were concentration-independent and relatively slow. Full recovery from TNP-ATP inhibition did not occur until ≥5 s after removal of the antagonist. Because of the slow off-kinetics of TNP-ATP, full competition with α,β-meATP for receptor occupancy could be seen only after both ligands had reached a steady-state condition. It is proposed that the slowly desensitizing P2X_2/3 receptor allowed this competitive interaction to be observed over time, whereas the rapid desensitization of other P2X receptors (P2X₃) may mask the detection of competitive inhibition by TNP-ATP.