Ford Kristen K, Matchett Michele, Krause James E, Yu Weifeng
Department of Electrophysiology, Neurogen Corporation, 35 Northeast Industrial Road, Branford, CT 06405, USA.
J Pharmacol Exp Ther. 2005 Oct;315(1):405-13. doi: 10.1124/jpet.105.088070. Epub 2005 Jul 13.
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 alphabeta-methylene ATP (alphabeta-meATP)-evoked P2X3 responses in a concentration-dependent manner (IC50 = 0.6 +/- 0.1 microM). IP5I effectively inhibited P2X3 currents when pre-exposed to desensitized but not unbound receptors. Furthermore, IP5I equally blocked 1 and 10 microM alphabeta-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 microM alphabeta-meATP significantly more than the one evoked by 10 microM alphabeta-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 alphabeta-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.
P2X3嘌呤能受体主要表达于背根神经节（DRG）神经元中，在痛觉中发挥重要作用。目前正在寻找P2X3特异性拮抗剂以改善多种适应症中的疼痛。了解拮抗剂如何与P2X3受体相互作用有助于发现和开发P2X3特异性拮抗剂。我们使用全细胞膜片钳技术，在离体大鼠DRG神经元中研究了非竞争性拮抗剂P1, P5 - 二[肌苷 - 5']五磷酸（IP5I）在P2X3受体上的活性，并与研究充分的竞争性拮抗剂TNP - ATP进行了比较。IP5I以浓度依赖性方式阻断αβ - 亚甲基ATP（αβ - meATP）诱发的P2X3反应（IC50 = 0.6±0.1微摩尔）。当预先暴露于脱敏但未结合的受体时，IP5I有效抑制P2X3电流。此外，IP5I同样阻断1微摩尔和10微摩尔αβ - meATP诱发的电流，并且对这些电流的脱敏速率常数没有影响。这支持了IP5I作为非竞争性拮抗剂与P2X3受体脱敏状态相互作用的作用。相比之下，TNP - ATP对1微摩尔αβ - meATP诱发的电流的抑制作用明显大于对10微摩尔αβ - meATP诱发的电流的抑制作用。它还显著减慢了电流的脱敏速率常数。这些结果表明TNP - ATP作为竞争性拮抗剂，在P2X3激动剂结合位点与αβ - meATP竞争。这些发现可能有助于解释为什么IP5I选择性作用于P2X嘌呤受体的快速脱敏P2X1和P2X3亚型，而对缺乏快速脱敏构象状态的缓慢脱敏P2X2和P2X(2/3)亚型的效力要低得多。
