P2X3受体受Pirt调节的分子机制及其在痛觉中的作用研究

P2X3 receptors play important roles in the passing of neuropathic pain and inflammatory pain. It's a potential target for the treatment of pain. Therefore, It's of great important to find and understand the intracellular molecules which can modulate the P2X3 receptor. Pirt is a kind of pain associated membrane protein which is specifically distributed in sensory neurons (nociceptors) of DRG. The Pirt-/- mice showed reduced responses to noxious stimuli. There is no report about the other functions of Pirt besides pain and itching. At the molecular level, the only thing that we known is that Pirt regulates the activity of TRPV1 channels in a C-terminal-dependent manner. Our previous study found that Pirt inhibited P2X3 current. The N-terminal of Pirt involved in this inhibition, but not the C-terminus. Biochemical experiments showd that Pirt co-immunoprecipited with P2X3 and the amount of membrane P2X3 protein decreased. In this project, we planned to study the mechanism of the interaction between Pirt and P2X3, to find the specific binding sites, to understand whether such interaction is involved in the modulation of pain, to determine whether the interference with such interaction can influence the development of pain, and tried to provide a new way to the treatment of pain.

P2X3受体在神经病理性痛和炎性痛中具有重要意义，是治疗疼痛的重要潜在靶点。寻找并认识可以调节P2X3受体的胞内分子，对指导疼痛的治疗具有重要意义。在DRG痛觉感受神经元上特异分布着一种与疼痛相关的膜蛋白分子Pirt。Pirt敲除后的小鼠表现出对伤害性刺激的反应减弱。除了疼痛和瘙痒以外，尚未见其他关于Pirt分子功能的报道。在分子水平，只知道Pirt可以通过C末端依赖的方式调节TRPV1通道活性。我们的前期研究发现，Pirt分子能够抑制P2X3的电流。Pirt的N端参与了这种抑制作用，而与C端无关。生化实验表明，Pirt与P2X3能发生免疫共沉淀，并且两者共表达时，胞膜P2X3蛋白量下降。本项目计划阐明Pirt与P2X3发生相互作用的分子机制，寻找具体的结合位点，判断这种相互作用是否参与调节疼痛，干预这种相互作用能否影响疼痛的发生发展，从而为疼痛的治疗提供新的思路。

Pirt是一种只表达在外周神经元上的穿膜蛋白。然而，Pirt在中空内脏里的生理和病理作用还不清楚。本研究中，我们发现Pirt缺失的小鼠膀胱活动过度活跃。Pirt缺失小鼠背根神经节神经元上α,β-meATP诱导的电流密度也显著增强。Pirt与P2X3受体共表达在膀胱神经纤维上，并且共表达的Pirt可以显著减少P2X3的电流。Pirt通过N端的14个氨基酸残基与P2X3发生相互作用。TAT穿膜肽融合的Pirt N端14小肽（Pirt(N14)）就足以抑制膀胱DRG神经元上的P2X3活性，也可以缓解Pirt敲除小鼠的膀胱过度活跃。用CYP处理小鼠（一种常用的膀胱过度活跃模型），会导致膀Pirt表达下降。重要的是，给予Pirt(N14)会减少CYP处理小鼠的膀胱收缩频率，回复排尿体积。因此，我们的结果表明Pirt是膀胱发挥功能过程中调节P2X3的一种内源性的分子。