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NEUROPHARMACOLOGY

The P2X₃ Antagonist P¹, P⁵-Di[inosine-5'] Pentaphosphate Binds to the Desensitized State of the Receptor in Rat Dorsal Root Ganglion Neurons

Departments of Electrophysiology (K.K.F., M.M., W.Y.) and Biochemistry and Molecular Biology (J.E.K.), Neurogen Corporation, Branford, Connecticut

P2X₃ purinergic receptors are predominantly expressed in dorsal root ganglion (DRG) neurons and play an important role in pain sensation. P2X₃-specific antagonists are currently being sought to ameliorate pain in several indications. Understanding how antagonists interact with the P2X₃ receptor can aid in the discovery and development of P2X₃-specific antagonists. We studied the activity of the noncompetitive antagonist P¹, P⁵-di[inosine-5'] pentaphosphate (IP₅I) at the P2X₃ receptor, compared with the well studied competitive antagonist TNP-ATP, using a whole-cell voltage-clamp technique in dissociated rat DRG neurons. IP₅I blocked -methylene ATP (-meATP)-evoked P2X₃ responses in a concentration-dependent manner (IC₅₀ = 0.6 ± 0.1 µM). IP₅I effectively inhibited P2X₃ currents when pre-exposed to desensitized but not unbound receptors. Furthermore, IP₅I equally blocked 1 and 10 µM -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 P2X₃ receptor. In contrast, TNP-ATP inhibited the current evoked by 1 µM -meATP significantly more than the one evoked by 10 µM -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 -meATP at the P2X₃ agonist binding site. These findings may help to explain why IP₅I acts selectively at the fast-desensitizing P2X₁ and P2X₃ subtypes of the P2X purinoceptor, while having much less potency at slow-desensitizing P2X₂ and P2X_2/3 subtypes that lack the fast desensitized conformational state.

Address correspondence to: Dr. Weifeng Yu, Neurogen Corporation, 35 Northeast Industrial Road, Branford, CT 06405. E-mail: wyu{at}nrgn.com