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.
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.
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