东亚钳蝎的TRP通道基因克隆与功能鉴定

The transient receptor potential (TRP) channels are widely distributed and emerging as a multifunctional set of membrane proteins involved a vast array of cellular processes and respond to a large number of physical and chemical stimuli, which involve them with sensory cell physiology. Living things must sense and respond to environmental changes. Scorpions known as ‘living fossils’ have existed on the earth for over than 400 million years and are adapted to have survived extreme climate changes. To date, whether TRP genes also express in scorpions and play essential roles in sensory transduction for scorpions still remain elusive. Based upon the large novel scorpion ion channel sequence informaiton from the genomic and transcriptomic data of Chinese Mesobuthus martensii achieved in the past years, we have successfully cloned five full length TRP genes coding for TRP like channels from Chinese scorpion, denoted as MmTRP1-5 channels. Our preliminary data demonstrated that one of them, MmTRP-1 channel, can be activated by camphor and menthol, respectively, when transiently expressed in HEK293 cells. Furthermore, MmTRP-1 channel mediates cationic currents with relatively high selectivity for Ca2+ over Na+. Moreover, MmTRP-1 channel displays a unique property by which the repeated stimulation causes successive increase in its activity, which is similar to that of thermal TRPV3 channel in mammalian cells. Based on the current advances, we focus this research on the identification and functional characterization of all five TRP genes already cloned from Chinese scorpion. The project aims to elucidate their activation and ion selectivity of the MmTRP1-5 channels, as well as to find out the certain stimuli that can gate the channels and what species of ion can pass through them. In addition, we will further explore the physiological functions mediated by the activation of MmTRP channel in the scorpion, especially in the sensing of environment. Since TRP channels play important roles in sensory physiology, this proposed research is therefore critical important for further understanding the TRP channel functions in detecting environmental changes.

瞬时受体势通道（TRP通道）分布广泛，响应种类多样的化学性和机械性刺激，在生物机体对外界环境的感知中发挥重要作用。感受外界环境变化的能力对生物体来说是相当重要的。被称为活化石的蝎子在地球上历经各种极端气候变迁已存续了4亿多年，那么作为环境感觉受体的TRP通道是否在蝎子中表达，并在蝎子对外界环境的“趋利避害”感知过程中发挥功能？基于课题组前期获得的我国东亚钳蝎基因组和转录组数据库中的离子通道序列信息，申请人前期成功地在东亚钳蝎中克隆得到五个具有完整序列的TRP基因，预实验结果表明其中的MmTRP-1通道可以分别被樟脑和薄荷醇激活，能够通透多种阳离子，并对Ca2+有较高的选择通透性。在此基础上，本研究旨在进一步对前期所获得的五个蝎TRP通道的功能进行鉴定，通过对这些蝎TRP通道的激活特性与离子选择性通透能力的阐释，进而探讨TRP通道在蝎子中的功能，所取得的研究结果将丰富人们对TRP功能的认识。

研究背景：瞬时受体势通道（TRP通道）广泛分布于神经和非神经组织中，响应细胞内外各种刺激，在生物机体对外界环境的感知中发挥重要作用。被称为活化石的蝎在地球上分布广泛，在地球上历经各种极端气候变迁已存续了4亿多年，该研究课题旨在探讨作为环境感受器的TRP通道是否在蝎子中表达，并在蝎子对外界环境的“趋利避害”感知过程中发挥功能？基于TRP通道在生理与病理中的重要作用，因此对其功能的调节研究也具有重要意义。..主要研究内容：本课题进一步对前期从中国东亚钳蝎中克隆得到的mmTRPg通道基因的功能进行鉴定，并探明了该通道在蝎的化学感受中的作用。此外，在实验研究执行过程中我们还对在温度感受中发挥重要作用的TRPV1和TRPV3通道的功能调节进行了研究。..研究结果：在本项自然科学基金的资助下，我们取得了如下重要进展：（1）完成了首个克隆自蝎的mmTRPg通道功能鉴定的研究，通过大规模筛选发现了多个能够激活通道的药物，这些药物能够引起蝎子的化学趋避反应，mmTRPg通道是一种对二价阳离子具有较高选择性通透的阳离子通道。此外还发现常见的杀虫剂和除草剂同样能够激活mmTRPg通道，而高浓度的药物能将蝎子致死。（2）将先进的光片显微成像技术与膜片钳电生理技术结合，实时跟踪观察了融合表达有pH-luorin GFP的TRPV1通道蛋白在不同刺激时的动态变化，结果表明，TRPV1通道脱敏中伴随有囊泡的内吞与胞吐一系列蛋白转运循环过程，高强度刺激时所引起的蛋白内吞很难在短时间里重新回到细胞膜上，揭示了TRPV1通道“记忆性”脱敏发生的机理。（3）发现并揭示了电压门控型Kv1.3通道在抗病毒反应中的重要作用。（4）分别阐明了药物1,4-Dioxane和Dyclonien对热敏TRPV1和TRPV3通道的调节作用及分子机理。..关键数据及其科学意义：研究结果揭示了TRP通道基因的保守性及其在环境感受中的重要作用，解析了TRPV1通道“记忆性”脱敏发生的机理，并阐明了药物对两个TRP通道的调节机制。光片显微成像技术与膜片钳电生理记录技术的成功整合将极大地促进类似膜离子通道蛋白转运相关研究的开展。