Store-operated Ca2+ influx & iPLA2 in vascular SMC

描述（由申请人提供）：我的长期目标是确定容量性Ca 2+内流的关键要素，并建立血管平滑肌细胞（SMC）中天然钙池操纵阳离子（SOC）通道调节的分子机制。最近，在建立了在激动剂诱导的平滑肌收缩和一氧化氮诱导的平滑肌松弛中钙池操作的（电容性）Ca 2+内流的生理重要性之后，我们成功地表征了一种新的小电导（3 pS）SOC通道，该通道负责SMC中的电容性Ca 2+内流。我们还确定了钙内流因子（CIF）激活这种天然SOC通道。尽管这种存储操作的途径非常重要，CIF诱导的SOC通道激活的分子机制仍然未知。我们最近的研究导致了全新的和非常有趣的结果，这些结果强烈表明，以前从未与该过程相关的Ca 2+非依赖性磷脂酶A2（iPLA 2）可以在SOC通道的激活和血管收缩中发挥关键作用。我们发现，抑制iPLA 2的表达和/或功能活性可防止Ca 2+内流的激活，并损害激动剂诱导的收缩。我们的初步数据表明，SOC通道的生理激活/抑制可以通过以膜界定的方式将抑制性CaM从iPLA 2置换/结合到iPLA 2来模拟，并且CIF可以将抑制性CaM从iPLA 2置换，导致其激活。我们也有证据表明iPLA 2活性的溶血磷脂产物可以激活SOC通道。本论文的总体目标是建立iPLA 2在容性Ca 2+内流途径和血管收缩中的新作用，并确定CIF诱导的SOC通道的iPLA 2依赖性激活的分子机制。 该提议的假设是iPLA 2是容量性Ca 2+内流和血管收缩的新分子决定因素，并且SOC通道激活是膜界定的CIF诱导的抑制性CaM从iPLA 2（其位于靠近SOC通道的质膜中）置换的结果。这将使用电生理学、分子、生物化学、成像和生理学方法对单个SOC通道、全细胞电流、血管SMC中的细胞内Ca 2+、iPLA 2表达和活性以及完整血管的收缩性水平进行测试。所有这些方法都已在实验室建立并成功应用。该模型和拟议研究的可行性得到了广泛的初步数据的充分支持。本提案的具体目标是：目标1。建立iPLA 2作为一种新的决定因素，在调控商店经营的渠道和血管收缩。我们将：确定iPLA 2是SOC通道的钙库依赖性激活和容量性Ca 2+内流的绝对必需的，确定iPLA 2在血管收缩中的新作用，确定iPLA 2的哪些特异性亚型参与钙库操纵的途径，确定iPLA 2在SMC中的定位。目标二。明确平滑肌细胞中钙库操纵通道的iPLA 2依赖性激活的分子机制。 我们将测试CIF诱导的iPLA 2和CaM介导的SOC通道激活的新模型中的所有步骤，并将确定iPLA 2是否可以被CIF激活，并通过消耗SMC中的Ca 2+储存，建立iPLA 2的CaM依赖性调节和SMC中SOC通道的CaM依赖性调节之间的直接相关性，测试CIF从iPLA 2置换CaM的能力，及其与CIF诱导的商店运营渠道激活的相关性，并确定iPLA 2的哪些特定产品激活本地商店运营渠道。

DESCRIPTION (provided by applicant): My long-term goal is to define the key elements in capacitative Ca2+ influx, and to establish the molecular mechanism of regulation of native store-operated cation (SOC) channels in vascular smooth muscle cells (SMC). Recently, after establishing the physiological importance of store-operated (capacitative) Ca2+ influx in agonist-induced contraction and nitric oxide-induced relaxation of SMC, we successfully characterized a novel small conductance (3 pS) SOC channel that is responsible for capacitahve Ca2+ influx in SMC. We also established that calcium influx factor (CIF) activates this native SOC channel. In spite of the tremendous importance of this store-operated pathway, the molecular mechanism of CIF-induced activation of SOC channels remains unknown. Our recent studies resulted in totally novel and very intriguing results which strongly suggest that Ca2+-independent phospholipase A2 (iPLA2), which has never been linked to this process before, can play a crucial role in activation of SOC channels and vascular contraction. We found that inhibition of expression and/or functional activity of iPLA2 prevents activation of Ca2+ influx, and impairs agonist-induced contraction. Our preliminary data showed that physiological activation/inhibition of SOC channels could be mimicked by displacement/association of inhibitory CaM from/to iPLA2 in membrane-delimited fashion, and that CIF could displace inhibitory CaM from iPLA2 resulting in its activation. We also have evidence that lysophospholipid products of iPLA2 activity can activate SOC channels. The overall goal of my proposal is to establish the novel role of iPLA2 in capacitative Ca2+ influx pathway and vascular contraction, and to determine the molecular mechanism of CIF-induced iPLA2- dependent activation of SOC channels. The hypothesis of this proposal is that iPLA2 is a novel molecular determinant of capacitative Ca2+ influx and vascular contraction, and that SOC channel activation is a result of membrane delimited CIF-induced displacement of inhibitory CaM from iPLA2, which is located in plasma membrane close to SOC channel. This will be tested using electrophysiological, molecular, biochemical, imaging and physiological approaches on the level of single SOC channels, whole-cell currents, intracellular Ca2+, iPLA2 expression and activity in vascular SMC, as well as contractility of intact blood vessels. All these methods are established and successfully used in Pl's lab. The feasibility of the model and proposed studies are fully supported by extensive preliminary data. Specific aims of this proposal are : Aim 1. To establish iPLA2 as a novel determinant in regulation of store-operated channels and vascular contraction. We will: Establish that iPLA2 is absolutely required for store-dependent activation of SOC channels and capacitative Ca 2+ influx, establish the novel role of iPLA2 in vascular contraction, determine which specific isoforms of iPLA2 are involved in store-operated pathway, and determine the location of iPLA2 in SMC. Aim 2. To define the molecular mechanism of iPLA2-dependent activation of store-operated channels in SMC. We will test all the steps in our novel model of CIF-induced iPLA2- and CaM-mediated activation of SOC channels, and will determine if iPLA2 can be activated by CIF, and by depletion of Ca 2+ stores in SMC, establish direct correlation between CaM-dependent regulation of iPLA2 and CaM-dependent regulation of SOC channels in SMC, test the ability of CIF to displace CaM from iPLA2, and its correlation with CIF-induced activation of store- operated channels, and determine which specific product(s) of iPLA2 activate native store-operated channels.