TRPV1通道一种新的失敏和功能调控机制：钙蛋白酶calpain介导的的剪切/降解作用

TRPV1 is a key molecule involved in the thermosensation and heat hyperalgesia. A remarkable feature of TRPV1 channel is the calcium-dependent desensitization. Calcium-dependent calpain protease is an important calcium effecter, which might contribute to TRPV1 desensitization and functional regulation. However, no data is available about the potential role of calpain in the functional regulation of TRPV1. Our preliminary data indicate that calpain is activated following the activation of TRPV1 by capsaicin. One of the cleavage sites of TRPV1 by calpain is located in the distal C-terminus. In the N-terminus of TRPV1, there is a concensus PEST (proline-glutamate-serine-threonine) motif for cleavage by calpain. In this project, we suppose the hypothesis "calpain is activated following the activation of TRPV1. Subsequently, calpain cleaves and degrades TRPV1, which participates in TRPV1 desensitization and functional regulation. This process constitutes a feedback regulatory pathway of TRPV1." We will prove the above hypothesis using multiple methods, including biochemical, proteomics, electrophysiological and behavioral tests, and attempt to identify the cleavage site and elucidate the functional effects of calpain-mediated cleavage of TRPV1. Our final goal is to construct the cell-permeable Tat-peptide to relieve pain through competitively inhibiting the phosphorylation of TRPV1, which is based on the inference that the phosphorylational modification of TRPV1 during pathological pain can interrupt the cleavage of TRPV1 by calpain.

TRPV1是介导热感受和热痛敏形成的关键分子，具有显著的钙依赖性失敏现象。钙蛋白酶 calpain是一种重要的钙效应分子，可能参与TRPV1通道失敏和功能调控，但尚未见相关文献报道。课题组预实验结果揭示，辣椒素激活TRPV1可活化calpain并发挥对TRPV1的剪切/降解作用，其中一个剪切位点近C端末尾，在通道N端还存在calpain底物偏好PEST基序。本项目拟围绕假说“TRPV1激活可活化calpain，calpain继而发挥对TRPV1的剪切/降解作用，由此参与通道失敏和功能调控，该过程构成TRPV1通道的一条反馈调节通路”，综合利用生物化学、蛋白组学、电生理学和行为学等多种方法，从离体细胞和在体组织水平，阐明calpain对TRPV1的剪切和功能调控作用。最终拟基于慢性痛情况下，TRPV1的磷酸化修饰可干扰calpain对其发挥作用的推论，设计Tat磷酸化干扰肽，用于热痛敏的治疗。

Ca2+通透的TRPV1通道是介导伤害性热感受和热痛敏形成的关键分子。钙蛋白酶calpain是一种重要的钙效应分子，可能参与TRPV1通道的功能调控，但尚未见相关文献报道。本项目综合利用生物化学、Fret和质谱检测等多种方法，证明辣椒素激活TRPV1可活化calpain并发挥对TRPV1的剪切/降解作用，并鉴定出其中一个剪切位点位于C端第819-820位氨基酸之间，由此形成的TRPV1功能截短体的功能还在鉴定中。受疫情影响，该部分工作尚未完全结束，正在加紧完成中。. 项目研究还发现钙感受蛋白KChIP3氨基端31-50位氨基酸与TRPV1胞内N端和C端均有直接的结合，并可抑制TRPV1膜表达。TAT-31-50穿膜肽可显著抑制TRPV1通道介导的Ca2+内流，减轻炎性痛模型大鼠热痛敏行为，改善其异常步态。该项研究揭示了KChIP3一种新的作用蛋白——TRPV1，证明KChIP3的外周镇痛作用依赖于其对TRPV1功能的抑制，同时，构建了一种具有镇痛作用的穿膜肽TAT-31-50，具有潜在的临床应用价值。上述工作发表在J Neurosci (2018)杂志上。. 此外，项目对于Kcnip3全基因敲除大鼠的研究还发现，这些大鼠表现出更显著的痛行为表现，提示KChIP3在体主要发挥抑制疼痛的作用，并且这些大鼠表现出更显著的痛相关厌恶情绪、更高的焦虑和抑郁水平，表明KChIP3参与负性情绪加工，提示KChIP3可能在脑水平影响痛觉信息加工。上述工作发表在Front Mol Neurosci (2019)杂志上。. 在本项目资助下，项目负责人作为通讯作者，发表SCI论文2篇，中文论文1篇，另有中文综述1篇（已接收）；作为第一作者，发表中文综述1篇。项目负责人在国内学术会议报告3次，培养（含联合培养）硕士研究生3名。