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.