Store Operated Calcium Channels Function in Vascular Smooth Muscle

该项目旨在了解由STIM蛋白介导的Ca[2+]的独特特征如何控制血管平滑肌细胞（VSMC）的增殖。Ca[2+]信号不仅在控制血管收缩，而且在调节平滑肌细胞生长和增殖中起着至关重要的作用。这项工作研究了两个关键蛋白STIM1和STIM2的功能，它们感知VSMCS SR管腔内Ca[2+]的变化，并通过高度协调的易位过程，进入SR和PM之间的小的特化连接。STIM蛋白直接激活PM中的Ca[2+]通道，从而控制Ca[2+]进入VSMCS。该项目有两个具体目的：目的1：研究STIM1和STIM2蛋白在VSMCs中介导Ca[2+]信号中的不同功能作用。这些研究将验证SM-STIM1-KO动物VSMC中STIM2表型具有“Ca[2+]特征反应”的假设，这在介导VSMC增殖的明显生理差异中很重要。验证这一假设的实验方法采用活细胞成像、显性负通道蛋白的表达、电生理学和新型Ca[2+]探针相结合的方法来评估stim介导的Ca[2+]信号在VSMCs中的功能作用，并检查：(a) SM-STIM1- KO小鼠VSMCs中Ca[2+]信号的时间Ca[2+]特征。(b) SM-STIM1-KO小鼠VSMCs中Ca[2+]信号的空间Ca[2+]特征；(c)腔内SR水平如何反映stim介导的VSMCs中Ca[2+]特征。目的2：确定stim诱导的Ca[2+]信号反应如何调节VSMCs的增殖途径。需要验证的假设是stim特异性Ca[2+]进入信号通过钙调神经磷酸酶/NFAT轴控制基因表达和增殖性VSMC反应。目的是确定(a) STIM蛋白如何控制增生性VSMC中钙调磷酸酶NFAT通路组分的表达。;(b) (b)刺激有丝分裂时，stim介导的Ca[2+]信号发生；(c) STIM蛋白如何控制NFAT易位/基因表达途径。

The project aims to understand how unique signatures of Ca[2+] mediated by STIM proteins control the proliferation of vascular smooth muscle cells (VSMC). Ca[2+] signals play a crucial role in not only controlling vascular contraction but also in regulating the growth and proliferation smooth muscle cells. The work examines the function of two crucial proteins, STIM1 and STIM2, that sense changes in the Ca[2+] within the SR lumen of VSMCS, and through a highly coordinated translocation process, move into small specialized junctions between the SR and PM. STIM proteins directly activate Ca[2+] channels in the PM and hence control Ca[2+] entry into VSMCS. The project has two specific aims: Aim 1: To examine the distinct functional roles of STIM1 and STIM2 proteins in mediating Ca[2+] signals in VSMCs. These studies will test the hypothesis that the STIM2 phenotype in VSMCs from SM-STIM1-KO animals has a ¿Ca[2+] signature response¿ important in mediating distinct physiological differences in VSMC proliferation. The experimental approach to test this hypothesis utilizes a combination of live cellular imaging, expression of dominant negative channel proteins, electrophysiology, and novel Ca[2+] probes to assess the functional roles of STIM-mediated Ca[2+] signals in VSMCs and examines: (a) the ¿temporal Ca[2+] signature¿ of Ca[2+] signals in VSMCs from SM-STIM1- KO mice. (b) the ¿spatial Ca[2+] signature¿ of Ca[2+] signals in VSMCs from SM-STIM1-KO mice: and (c) how luminal SR levels reflect the STIM-mediated Ca[2+] signature in VSMCs. Aim 2: To determine how STIM-induced Ca[2+] signature responses regulate proliferative pathways in VSMCs. The hypothesis to be tested is that STIM-specific Ca[2+] entry signals control gene expression and proliferative VSMC responses through the calcineurin/NFAT axis. The aims are to determine (a) how STIM proteins control expression of components of the calcineurin NFAT pathway in proliferative VSMC. ; (b) (b) STIM-mediated Ca[2+] signaling occurs during mitogenic stimulation; (c) how STIM proteins control the NFAT translocation/gene expression pathway.