植物细胞水分变化感应分子CSCs的分离、鉴定和调控机制研究

Water is the origin of life. Cytosolic calcium elevation is one of the earliest responses of plant cells to water changes，However, none of the primary messengers or receptors has been found to respond directly to this signal. In an attempt to identify this kind of molecule, we used heterologous expression of Arabidopsis CDS genes in Chinese Hamster Ovary (CHO) cells and screened membrane proteins for osmosensitive conductance. These screens led to the identification of Arabidopsis CSC1 (Calcium permeable Stress-gated cation Channel 1) that cause the intracellular Ca2+ concentration promptly rising by water changes. Based on these results, we try to determine the receptor-channel electrophysiological characterization of CSCs, including ion selectivity, rectification and water-change gating of the property, to identify their functional domains for osmotic sensing and the Ca2+-messenger conductance, In order to further investigation the regulation of CSCs, we will isolate the interacting factors of this protein and reconstitute the partners in vitro. Morever, by employing anti-genetic and trangentic approaches to analyze the phenotype of cscs mutants and the physiological function of CSCs, especially the rapid reponsiveness to water changes.

细胞感应水分变化是生命的基本生理过程，寻找水分胁迫或干旱信号接收和传递的感应或受体分子，一直是人们追求的目标。从胞内钙离子上升、感应水分胁迫的原初反应出发，利用膜蛋白CDS文库，我们筛选出对渗透变化快速反应的多个细胞株，进而克隆了AtCSC1（Calcium permeable Stress-gated cation Channel 1）,经研究现已发现CSCs可介导水分变化门控的第二信使离子内流,但其感应、通道特征、生理功能和调控途径仍不清楚。本课题以此为切入点，深入解析CSCs作为水分感应通道分子的特性和结构元件，分析该蛋白的通道选择性、渗透门控特性等，鉴定出接受水分变化刺激的结构域和导致钙离子信使信号传递的结构域，探索分离出该蛋白的相互作用或调节分子，重组调控途径，结合反向遗传学和转基因的方法在体内研究CSCs突变体的表型及生理功能，理解作CSCs为水分变化原初信号接收分子的特性.

接收水分胁迫或干旱信号的受体分子一直是人们追求的目标，本项目首先筛选出对渗透变化快速反应的多个细胞株，分离、发现了对水分胁迫快速反应的AtCSC1，属于未知功能蛋白家族（DUF221），进一步研究发现这一家族成员不仅能感应传递高渗信号，也能感应其他的机械刺激或膜形变信号，编码第二信使钙离子波峰。随后分析了人类和酵母的同源蛋白HsTM63C和ScYLR241W的电生理学特性，它们与AtCSC1类似，也是电压依赖的阳离子非选择性通道，且具有渗透或机械刺激门控特性。同时解析了该家族蛋白的功能结构域：其中接受水分变化或机械刺激的结构域为CSC-N端，而介导钙离子信号传递的结构域为C端（6TM-C-Terminal），分离出了该类蛋白的相互作用分子如：CAMK、Clp6和RING-finger等，体外重建调控途径的工作正在进行；获得了 CSCs多个单突变体和不同的多突变体，例如： mcsc-like2（tmem63B）导致小鼠的耳聋。总之，圆满完成了本课题的研究目标，并为进一步研究开辟了新的方向。