Mechanism of nociception : Structure-function relationship of capsaicin receptor and analysis of its regulation mechanism

与感染、炎症或缺血相关的组织损伤会产生一系列化学介质，激活或敏化伤害性感受器终端，引起损伤部位的疼痛。这种前痛觉反应的一个重要组成部分是从不同类型的细胞释放的ATP。细胞外的三磷酸腺苷刺激附近感觉神经的伤害性末梢，引起痛感。为探讨代谢型P2Y受体是否参与VR1介导的伤害性反应，观察了细胞外ATP对HEK293细胞和大鼠背根神经节神经元VR1表达的影响。在表达VR1的细胞中，胞外ATP通过PKC依赖的途径激活代谢性P2Y1受体，从而增加辣椒素或质子诱发的电流。在三磷酸腺苷存在的情况下，Vr1激活的温度阈值从42℃降低到35℃，从而使正常的非痛性热刺激(即正常体温)能够激活vr1。这代表了一种新的机制，ATP可能通过不同于P2X受体激活的途径引起疼痛。接下来，必须解决的是，VR1是否被PKC直接磷酸化，如果是，哪些氨基酸残基参与了磷酸化。在表达VR1的细胞中，生物化学证明了在应用PMA时VR1的直接磷酸化。用谷胱甘肽S转移酶融合蛋白与vr1胞浆片段进行体外激酶分析表明，vr1的第一个胞内环和羧基末端都被PKCε磷酸化。对VR1中16个可能的磷酸化位点中Ser或Thr残基被Ala取代的点突变的膜片钳分析表明，两个Ser残基参与了PMA或ATP诱发的电流的增强。这两个位点将是开发调节VR1功能的物质的有希望的靶点，从而减轻疼痛。

Tissue damage associated with infection, inflammation, or ischemia, produces an array of chemical mediators that activate or sensitize nociceptor terminals to elicit pain at the site of injury. An important component of this pro-algesic response is ATP released from different cell types. Extracellular ATP excites the nociceptive endings of nearby sensory nerves, evoking a sensation of pain. To address whether metabotropic P2Y receptors are involved in VR1-mediated nociceptive responses, the effects of extracellular ATP on VR1 expressed in HEK293 cells and rat DRG neurons were examined. In cells expressing VR1, extracellular ATP increased the currents evoked by capsaicin or protons through activation of metabotropic P2Y_1 receptors in a PKC-dependent pathway. In the presence of ATP, the temperature threshold for VR1 activation was reduced from 42 ℃ to 35 ℃, such that normally non-painful thermal stimuli (i.e. normal body temperature) were capable of activating VR1. This represents a novel mechanism through which ATP might cause pain in a pathway distinct from the activation of P2X receptors.Next, it has to be addressed whether VR1 is directly phosphorylated by PKC and if so which amino acid residues are involved in the phosphorylation. Direct phosphorylation of VR1 upon application of PMA was proven biochemically in cells expressing VR1. An in vitro kinase assay using GST fusion proteins with cytoplasmic segments of VR1 showed that both the first intracellular loop and carboxy terminal of VR1 were phosphorylated by PKCε. Patch-clamp analysis of the point mutants where Ser or Thr residues were replaced with Ala in the total 16 putative phosphorylation sites in VR1 showed that two Ser residues were involved in the potentiation of the currents evoked by either PMA or ATP. These two sites would be promising targets for the development of substance modulating VR1 function, thereby reducing pain.