Involvement of P2 receptors in the modulation of nociceptive afferent stimuli

P2X3受体在伤害性背根神经节(DRG)神经元中具有高选择性表达。因此，深入了解受体的结构和功能对于开发以前被证明在各种疼痛状态下有效的治疗药物的选择性拮抗剂具有极其重要的意义。这些实验将在转染野生型或突变型P2X3或TRPV1受体的HEK293细胞以及DRG神经元中进行。我们旨在研究以下问题：A.P2Y1和P2X3受体之间的相互作用。我们打算确定参与这种相互作用的第二信使途径。P2Y1受体最可能的转导机制包括Gq/11/磷脂酶C/PKC和Gq/11/磷脂酶C/IP3/Ca~2途径，或通过膜限制的抑制作用。我们将研究共同定位于同一DRG神经元的这些受体之间的负相互作用。C.核苷酸结合部位和糖基化部位。我们已经通过分子模拟确定了P2X3受体可能的激动剂结合部位和糖基化部位。现在我们打算用实验来证实我们的预测。D.P2X3受体的贩运。通过激光共聚焦扫描显微镜，我们将跟踪这些受体在细胞内的运输。

P2X3 receptors are expressed with high selectivity in nociceptive dorsal root ganglion (DRG) neurons. Therefore, an intimate knowledge of the receptor-structure and -function is of utmost significance for the development of selective antagonists previously shown to be effective as therapeutic agents in various pain states. The experiments will be performed at HEK293 cells transfected with wild-type or mutant P2X3 or TRPV1 receptors, and in DRG neurons. We aim at investigating the following questions: A. Interaction between P2Y1 and P2X3 receptors. We intend to identify the second messenger pathways involved in this interaction. The most likely transduction mechanisms of the P2Y1 receptor include the Gq/11/phospholipase C/PKC and the Gq/11/phospholipase C/IP3/Ca2+ pathways or a membrane delimited inhibition via the ß¿ subunits.B. Interaction between P2X3 and TRPV1 receptors. We will investigate the negative interactionbetween these receptors co-localized at the same DRG neurons. C. Nucleotide binding sites and glycosylation sites. We have identified by molecular simulation the possible agonist binding sites and the glycosylation sites of the P2X3 receptor. Now we intend to experimentally confirm our predictions. D. Trafficking of P2X3 receptors. By means of confocal laser scanning microscopy we will follow the intracellular trafficking of these receptors.