Store Operated Calcium Channels Function in Vascular Smooth Muscle

描述(申请人提供)：该项目旨在了解STIM蛋白介导的独特的钙信号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方面起着至关重要的作用，而且在调节平滑肌细胞的生长和增殖方面也起着至关重要的作用。这项工作研究了两种关键蛋白质STIM1和STIM2的功能，这两种蛋白质感知VSMCS SR管腔内钙离子的变化，并通过高度协调的易位过程进入SR和PM之间的小型专门化连接Stim蛋白直接激活质膜上的钙通道，从而控制钙离子进入血管平滑肌细胞。该项目有两个特定的目标：目标1：研究STIM1和STIM2蛋白在VSMC中介导钙信号的不同功能。这些研究将检验这一假设，即SM-STIM1-KO动物VSMCs中的STIM2表型在调节VSMC增殖的明显生理差异方面具有重要的“钙信号反应”。验证这一假说的实验方法利用活细胞成像、显性负通道蛋白的表达、电生理学和新型钙探针来评估STIM介导的钙信号在VSMC中的功能作用，并检查：(A)SM-STIM1-KO小鼠VSMCs中钙信号的“时间钙信号”；(B)SM-STIM1-KO小鼠VSMCs中钙信号的“空间钙信号”；以及(C)腔SR水平如何反映STIM介导的VSMC中的钙信号。目的：研究STIM诱导的钙信号反应对VSMC增殖途径的调节作用。需要检验的假设是，STIM特异的钙离子进入信号通过钙调神经磷酸酶/NFAT轴控制基因表达和增殖的VSMC反应。其目的是确定(A)STIM蛋白如何控制增殖的VSMC中钙调神经磷酸酶NFAT通路的表达；(B)STIM介导的钙信号发生在有丝分裂刺激过程中；(C)STIM蛋白如何控制NFAT转位/基因表达途径。

DESCRIPTION (provided by applicant): The project aims to understand how unique signatures of Ca2+ mediated by STIM proteins control the proliferation of vascular smooth muscle cells (VSMC). Ca2+ signals play a crucial role in not only controlling vascular contraction but also in regulating the growth and proliferation of smooth muscle cells. The work examines the function of two crucial proteins, STIM1 and STIM2, that sense changes in the Ca2+ 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 Ca2+ channels in the PM and hence control Ca2+ entry into VSMCS. The project has two specific aims: Aim 1: To examine the distinct functional roles of STIM1 and STIM2 proteins in mediating Ca2+ signals in VSMCs. These studies will test the hypothesis that the STIM2 phenotype in VSMCs from SM-STIM1-KO animals has a "Ca2+ 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 Ca2+ probes to assess the functional roles of STIM-mediated Ca2+ signals in VSMCs and examines: (a) the "temporal Ca2+ signature" of Ca2+ signals in VSMCs from SM-STIM1- KO mice; (b) the "spatial Ca2+ signature" of Ca2+ signals in VSMCs from SM-STIM1-KO mice; and (c) how luminal SR levels reflect the STIM-mediated Ca2+ signature in VSMCs. Aim 2: To determine how STIM-induced Ca2+ signature responses regulate proliferative pathways in VSMCs. The hypothesis to be tested is that STIM-specific Ca2+ 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) STIM-mediated Ca2+ signaling occurs during mitogenic stimulation; (c) how STIM proteins control the NFAT translocation/gene expression pathway.