伤害性感受器TRPV2通道的内源性调节机制研究

Nociceptive receptor TRPV2 channel is one member of the transient receptor potential (TRP) superfamily, sharing the common property of polymodal gating as many other TRP channels. An increasing number of studies have suggested that TRPV2 channels are involved in diverse cellular processes and play important roles in thermal pain sensation, chemosensation and mechanical sensation. To date, the researches on TRPV2 are slower than other TRP channels because of its reported activation by extremely high temperatures (>52℃), and as well as the scarcity of specific endogenous ligands or regulatory subunits and/or pharmacological drugs. In our preliminary experiments, we interestingly found that: 1) Ca2+ at physiological concentrations would not only cause the desensitization of TRPV2 channels, but also increase the sensitivities of TRPV2 to its agonist and thus enhance the channel activities. Moreover, our results revealed that TRPV2 also has a calcium-independent desensitization in addition to calcium-dependent desensitization; 2) Mg2+ within the physiological concentrations also remarkably enhanced TRPV2 channel functions, while Ba2+ and Zn2+ could respectively inhibit TRPV2 channel currents; 3) Through yeast two-hybrid experiments, we found several potential regulatory and accessory proteins interacting with TRPV2 channels in native tissues. One of them, KCNAB1 has been shown to interact with TRPV2 by our coimmunoprecipitation (CO-IP) analysis, and we further found that co-expression with KCNAB1 would increase cell surface expression levels of TRPV2. Based upon our preliminary data, this proposed research therefore aims to elucidate the effects and the mechanisms underlying upregulation of TRPV2 channel functions by Ca2+ and Mg2+ at physiological concentrations, respectively. Also, we will compare and demonstrate the differences between Ca2+-dependent and Ca2+-independent desensitization, and find out how to recover TRPV2 function from the desensitized state. In addition to clarify the protein-protein interaction of TRPV2 with potential interaction partners discovered in our previous yeast two-hybrid experiments, we will demonstrate how KCNAB1 interacts with TRPV2 channel protein and the mechanism underlying KCNAB1 increased cell surface expression levels of TRPV2 as well. Taken together, our research aims to define pathways that control and regulate the activities of TRPV2 channels. TRPV2 channel has been implicated in diverse physiological and pathophysiological processes, including thermo sensation, pain transduction, innate immunity and cancer. Therefore the proposed research will provide insightful information on regulating TRPV2 by endogenous factors and is critically important for a better understanding TRPV2 physiology.

伤害性感受器TRPV2通道功能多样，在机体的热痛感觉、化学刺激和机械刺激感觉等过程中发挥重要作用，但由于TRPV2极高的温度激活阈值（>52℃），加之目前缺乏专一的工具药物或调节亚基，而使得对TRPV2通道门控和调节机制的研究相对滞后。而我们在前期预实验中发现了多种内源性因素均可以有效调节TRPV2通道功能，包括：1）生理浓度范围的Ca2+和Mg2+均能显著地增强TRPV2的活性，而Ba2+和Zn2+则对TRPV2起抑制作用；2）TRPV2兼具Ca2+依赖和Ca2+不依赖两种脱敏特性；3）通过酵母双杂交实验筛选得到多个与TRPV2互作的候选蛋白，初步结果显示其中的KCNAB1亚基通过与TRPV2互作而提高TRPV2通道的上膜量。基于前期发现，本研究旨在阐明上述内源性调节因素对TRPV2通道功能的调节机理，所获研究结果将有助于丰富内源性调控TRPV2活性的手段，促进TRPV2的生理功能研究。

研究背景：伤害性感受器TRPV2通道在机体中广泛分布，在不同的细胞组织中扮演不同的角色，与热感知、疼痛传导、天然免疫和肿瘤等生理病理过程均密切相关，但由于TRPV2极高的温度激活阈值（>52℃），加之目前缺乏专一的工具药物或调节亚基，而使得对TRPV2通道门控和调节机制的研究相对滞后。与TRPV2同处相同亚家族的辣椒素受体TRPV1通道在温度感知和疼痛传导上亦具有重要作用，其激动剂如辣椒素能够通过引起痛觉脱敏而起到镇痛作用，而TRPV1拮抗剂能有效缓解啮齿动物模型的炎症、关节炎和癌症的疼痛行为，因此TRPV1通道是有效控制疼痛的一个在体靶点。以TRPV1通道作为靶点的研究正在促进新型镇痛疗法的发展。基于TRPV2和TRPV1通道在生理与病理活动中的重要性，对它们的功能调节进行研究具有重要的科学意义和临床价值。天然免疫细胞和适应性免疫细胞中也表达了各种各样的离子通道，探讨离子通道在抗病毒感染免疫反应中的重要作用是生物医学研究领域的研究热点。..主要研究内容：在前期预实验结果的基础上，本研究旨在：1）阐明Ca2+、Mg2+等多种阳离子对TRPV2和TRPV1通道的调节机制；2）探明互作蛋白Flotillin-1和Kvb1分别对TRPV2和TRPV1通道功能的调节机制；3）解析SUMO化修饰调控TRPV1通道活性的作用机理；4）靶向TRPV通道的作用药物发现。..重要结果：在本项基金的资助下，我们取得了如下结果：（1）构建了TRPV1-pHluorin荧光探针，实现了活细胞中对TRPV1功能及通道蛋白循环转运的并行检测，阐明了TRPV1通道Ca2+依赖的“记忆性”脱敏机制;（2）发现并揭示了SUMO/deSUMO化动态修饰调控TRPV1通道介导的热敏疼痛的分子机理；（3）解析了Kv1.3通道在抗病毒感染免疫反应中的重要作用，综述了离子通道在免疫细胞和免疫应答反应中的功能研究进展；（4）发现并阐释了Flotillin-1和Kvb1分别通过与TRPV2和TRPV1通道蛋白直接互作而调控通道活性的机理；（5）完成了1,4-dioxane和dyclonine等药物对TRPV1和TRPV3通道功能能调控机理的研究。..关键数据及其科学意义：研究结果丰富了调控TRP通道功能的手段，为临床疼痛的诊疗提供了理论基础，并为相关药物设计提供了借鉴与新思路。

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