权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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

伤害感受机制：辣椒素受体的结构-功能关系及其调控机制分析

基本信息

批准号：
12670037
负责人：
TOMINAGA Makoto
金额：
$ 2.43万
依托单位：
Mie University (2001)University of Tsukuba (2000)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670037/
关键词：
Pain capsaicin receptor VR1 phosphorylation ATP P2Y_1 receptor PKC PKC プロトン

项目摘要

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.

与感染、炎症或缺血相关的组织损伤产生一系列化学介质，其激活或致敏伤害感受器末端以在损伤部位引起疼痛。这种促痛觉反应的一个重要组成部分是从不同细胞类型释放的ATP。细胞外ATP刺激附近感觉神经的伤害性末梢，引起疼痛感。为了解决代谢型P2Y受体是否参与VR1介导的伤害性反应，检测了细胞外ATP对HEK293细胞和大鼠DRG神经元中表达的VR1的影响。在表达VR1的细胞中，细胞外ATP通过激活PKC依赖的代谢型P2Y_1受体增加辣椒素或质子诱发的电流。在存在ATP的情况下，VR1激活的温度阈值从42 ℃降低至35 ℃，使得正常非疼痛性热刺激（即正常体温）能够激活VR1。这代表了一种新的机制，ATP可能通过一种不同于P2X受体激活的途径引起疼痛。接下来，必须解决VR1是否直接被PKC磷酸化，如果是这样，哪些氨基酸残基参与磷酸化。在表达VR1的细胞中，经生物化学证实PMA可直接磷酸化VR1。使用具有VR 1胞浆片段的GST融合蛋白进行的体外激酶测定显示，VR 1的第一个胞内环和羧基末端均被PKCε磷酸化。在VR1的16个磷酸化位点中，Ser或Thr残基被Ala取代的点突变体的膜片钳分析表明，两个Ser残基参与PMA或ATP诱发的电流增强。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。