The P2X3 antagonist P1, P5-di[inosine-5′] pentaphosphate binds to the desensitized state of the receptor in rat dorsal root ganglion neurons

P2X(3) purinergic receptors are predominantly expressed in dorsal root ganglion (DRG) neurons and play an important role in pain sensation. P2X(3)-specific antagonists are currently being sought to ameliorate pain in several indications. Understanding how antagonists interact with the P2X(3) receptor can aid in the discovery and development of P2X(3)-specific antagonists. We studied the activity of the noncompetitive antagonist P-1, P-5-di[inosine-5'] pentaphosphate (IP5I) at the P2X(3) receptor, compared with the well studied competitive antagonist TNP-ATP, using a whole-cell voltage-clamp technique in dissociated rat DRG neurons. IP5I blocked alpha beta-methylene ATP (alpha beta-meATP)-evoked P2X(3) responses in a concentration-dependent manner (IC50 = 0.6 +/- 0.1 mu M). IP5I effectively inhibited P2X(3) currents when pre-exposed to desensitized but not unbound receptors. Furthermore, IP5I equally blocked 1 and 10 mu M alpha beta-meATP-evoked currents and had no effect on the desensitization rate constant of these currents. This supports the action of IP5I as a noncompetitive antagonist that interacts with the desensitized state of the P2X(3) receptor. In contrast, TNP-ATP inhibited the current evoked by 1 mu M alpha beta-meATP significantly more than the one evoked by 10 mu M alpha beta-meATP. It also significantly slowed down the desensitization rate constant of the current. These results suggest that TNP-ATP acts as a competitive antagonist and competes with alpha beta-meATP at the P2X(3) agonist binding site. These findings may help to explain why IP5I acts selectively at the fast-desensitizing P2X(1) and P2X(3) subtypes of the P2X purinoceptor, while having much less potency at slow-desensitizing P2X(2) and P2X(2/3) subtypes that lack the fast desensitized conformational state.