Ion channels and calcium regulation in vascular SMC function

描述(申请人提供)：我们的长期目标是确定不同的钙离子进入途径在血管平滑肌细胞(SMC)功能中的分子机制和生理作用。这一建议是我们对血管SMC中存储操作通道(SOC)和钙内流(SOCE)的成功研究的合乎逻辑的继续。最近我们发现，相同的Orai1基因可以编码具有截然不同的选择性的通道，而ADAR1介导的RNA编辑可能是不同细胞类型中Orai1特性的一种分子机制。我们从原代培养的血管SMC中确定了Orai1mRNA中可能的RNA编辑位点，并证明了单点突变(模仿RNA编辑事件)可以将钙离子选择性的CRAC转化为CAT-SOC通道。我们还获得了证据表明，iPLA2b依赖的Orai1激活可能在原代血管SMC的增殖和迁移中起关键作用。我们的初步研究为我们的新建议提供了坚实的概念和实验基础，该建议旨在解决长期存在的关于血管SMC中CAT-SOC通道分子身份的争议，确定SMC迁移过程中新的钙离子进入机制，并为心血管疾病的治疗建立新的分子机制和靶点。我们的中心假设是，RNA编辑的Orai1编码CAT-SOC通道，在血管SMC迁移中发挥重要作用。我们建议使用我们的综合方法进行深入研究，包括高级分子、生化、成像、电生理和单个分子的功能表征，以及原代血管SMC和模型细胞系中的信号级联。所有的方法和方法在PI的实验室中都得到了成功的应用。大量的初步数据充分证明了拟议研究的可行性。我们提出：目的1.确定Orai1的RNA编辑的分子机制和后果。我们将测试Orai1是依赖ADAR1的RNA编辑的生物靶点的假设，将表征可以改变Orai1编码通道的选择性、糖基化和其他生物物理性质的RNA编辑事件，并确定编辑单个Orai1亚基如何影响Orai1四聚体的特性。目的2.鉴定原代血管SMC中天然CAT-SOC通道的分子结构。我们将测试假设，即RNA编辑的Orai1编码天然的CAT-SOC通道，并将表征ADAR1介导的RNA编辑和Orai1在原代血管SMC中的脱糖基化。目的3.建立Orai1参与SMC迁移的新的作用和分子机制：我们将确定iPLA2b和Orai1在SMC迁移中的作用和空间分布，并验证Orai1介导的钙内流参与局部黏附的形成和成熟，和/或促使SMC迁移的力量产生的假说。 公共卫生相关性：我们的长期目标是确定特定离子通道在调节血管平滑肌细胞(SMC)功能的钙稳态中的作用。该方案的目的是确定Orai1(一种特定的质膜蛋白)在编码天然钙离子内流通道中的作用，并确定它们在SMC迁移中的生理作用。这些研究的可行性得到了PI实验室广泛的初步数据和先进的专业知识的充分支持。

DESCRIPTION (provided by applicant): Our long term goal is to define molecular mechanism and physiological role of different Ca2+ entry pathways in vascular smooth muscle cell (SMC) function. This proposal is a logical continuation of our successful studies of the store-operated channels (SOC) and Ca2+ entry (SOCE) in vascular SMC. Recently we discovered that the same Orai1 gene can encode channels with profoundly different selectivity, and ADAR1-mediated RNA editing can be a molecular mechanism for versification of Orai1 properties in different cell types. We identified putative RNA editing sites in Orai1 mRNA from primary vascular SMC, and demonstrated that single point mutation (that mimics RNA editing event) can transform Ca2+ selective CRAC into cat-SOC channel. We also obtained evidence that iPLA2b-dependent activation of Orai1 may be crucial for proliferation and migration of primary vascular SMC. Our preliminary studies provided solid conceptual and experimental foundation for our new proposal that is posed to resolve the long lasting controversy about the molecular identity of cat-SOC channels in vascular SMC, identifynew Ca2+ entrymechanisminvolved in SMCmigration, andestablish new molecular mechanisms and targets for treatment of cardiovascular diseases. Our central hypothesis is that RNA-edited Orai1 encodes cat-SOC channels and plays important role in vascular SMC migration. We propose in-depth studies using our integrative approach that involves advanced molecular, biochemical, imaging, electrophysiological and functional characterization of individual molecules and signaling cascades in primary vascular SMC and model cell lines. All approaches and methods are successfully used in the PI's lab. The feasibility of proposed studies is fully justified by extensive preliminary data. We propose: Aim 1. To determine the molecular mechanism and consequences of RNA-editing of Orai1.. We will test hypothesis that Orai1 is a biological target for ADAR1-dependent RNA editing, will characterize RNA editing events that can change selectivity, glycosylation and other biophysical properties of Orai1-encoded channels, and determine how editing of individual Orai1 subunits affect the properties of Orai1 tetramers. Aim 2. To identify molecular organization of native cat-SOC channel in primary vascular SMC. We will test hypothesis that RNA-edited Orai1 encodes native cat-SOC channel and will characterize ADAR1-mediated RNA editing and de-glycosylation of Orai1 in primary vascular SMC. Aim 3. To establish the novel role and molecular mechanism of Orai1 involvement in migration of SMC: We will determine the role and spatial distribution of iPLA2b and Orai1 in migrating SMC and test the hypothesis that Orai1-mediated Ca2+ entry is involved in focal adhesion formation and maturation, and/or force production that enables migration of SMC. PUBLIC HEALTH RELEVANCE: Our long term goal is to define the role of specific ion channels in calcium homeostasis which regulates vascular smooth muscle cell (SMC) function. The goals of this proposal are to determine the role of Orai1 (a specific plasma membrane protein) in encoding native store-operated calcium influx channels, and to establish their physiological role in SMC migration. The feasibility of these studies is fully supported by extensive preliminary data and advanced expertise of PI's lab.