钙结合蛋白DREAM与TRPV1的相互作用及其对TRPV1通道功能调控的研究

It is generally accepted that TRPV1 is a key molecule involved in the development of peripheral sensitization during pathological pain states. As reported recently, TRPV1 also plays a critical role in pain modulation in the central nervous system. Analgesics targeting TRPV1 are promising. However, both TRPV1 agonists or antagnists have some side effects when they are used for pain relief. It might be a new strategy for the development of analgesics based on the regulatory proteins of TRPV1, which can interfere with the function of TRPV1. Our preliminary data indicate that DREAM (downstream regulatory element antagonist modulator), a Ca2+-binding protein highly expressed in neurons, associates with TRPV1 and inhibits the surface localization of TRPV1. These findings imply that DREAM might be a negative regulator of TRPV1. In the present project, we will investigate the interaction between DREAM and TRPV1, the regulatory role of DREAM in TRPV1 channel current, desensitization and surface expression and reveal the underlying mechanisms, using biological chemistry, molecular biological, electrophysiological and behavioral methods. More importantly, we will construct the transmembrane peptides according to the critical binding sites of DREAM and detect their effects on TRPV1 channel function and the possible analgesic effects. These findings are of great significance for our understanding of the regulatory mechanisms of TRPV1 and the development of analgesics.

TRPV1不仅是公认的参与病理性疼痛外周敏化的关键分子，近年来证实中枢TRPV1也发挥重要的痛觉调制作用。以TRPV1为靶点的镇痛药物前景良好，但现有TRPV1激动剂或拮抗剂在镇痛同时伴一定副作用而影响其在临床的使用。从TRPV1调节蛋白入手设计TRPV1功能干扰药物，或将是镇痛药物研发的新途径。本室预实验结果揭示神经元高表达的钙结合蛋白DREAM可与TRPV1结合，并抑制TRPV1膜表达，可能是TRPV1的负性调控蛋白。本项目拟在原有工作基础上，综合利用生物化学、分子生物学、电生理学和动物行为学等方法，对DREAM与TRPV1的相互作用及其对TRPV1通道电流、失敏和膜表达的调控和机制进行深入研究。更重要的是，我们拟根据DREAM与TRPV1结合的主要位点构建穿膜肽，观察其是否可抑制TRPV1功能并发挥镇痛作用。该研究对深入认识TRPV1调控机制，开发新型镇痛药物具有重要意义。

KChIP3/DREAM/Calsenilin（以下简称KChIP3）是一种神经系统高表达的多功能蛋白，隶属NCS（neuronal calcium sensor）家族。既往研究集中在KChIP3在中枢的功能，如本课题组在2010年首次报道KChIP3对NMDA受体的功能调控及其在缺血性脑损伤中的作用（Zhang Y et al. J Neurosci. 2010 Jun 2;30(22):7575-86）。而近些年来的多项研究显示KChIP3在背根神经节（dorsal root ganglion，DRG）有表达，提示其可能参与外周感觉信息加工，但详细机制尚不清楚。在本研究中，我们对KChIP3在外周DRG的功能进行了探讨。结果表明，KChIP3与伤害性热感受的关键分子TRPV1（transient receptor potential vanilloid 1）有共定位，并且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，可引起TRPV1膜表达下降和激动剂辣椒素诱发的钙内流减少。更重要的是，TAT序列引导的可穿膜肽TAT-31-50也可以显著抑制TRPV1通道介导的钙内流，经鞘内注射给药后可减轻完全弗氏佐剂（complete Freund‘s adjuvant，CFA）诱导炎症痛模型鼠热痛敏行为，改善异常步态。此外，为进一步确认TAT-31-50在外周的作用，我们经脚掌内注射给药，验证了TAT-31-50减缓辣椒素诱导疼痛大鼠自发痛和热痛敏行为的效应。由于在课题组通过CRISP/CAS9构建的kcnip3-/-大鼠中，仍然可以检测到TAT-31-50的镇痛效应，因此TAT-31-50抑制TRPV1受体功能的效应并不依赖于内源性KChIP3。总之，我们的研究揭示了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，本研究还构建了一种可发挥镇痛作用的穿膜肽TAT-31-50，具有潜在临床应用价值。本项目已发表标注资助SCI论文5篇，包括Exp Neurol, Neuropsychopharmacol, Neursci Bull, Nature Communi等，另有1篇已被J Neurosci接收，总影响因子37.85，发表中文综述2篇，参加国际学术会议2次，并均获优秀展板奖。