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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL054150-15
Application #
8264983
Study Section
Special Emphasis Panel (ZRG1-CVRS-C (02))
Program Officer
OH, Youngsuk
Project Start
1996-08-05
Project End
2014-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
15
Fiscal Year
2012
Total Cost
$431,550
Indirect Cost
$176,195
Name
Boston Medical Center
Department
Type
DUNS #
005492160
City
Boston
State
MA
Country
United States
Zip Code
02118
Bolotina, Victoria M (2012) Orai1, STIM1, and iPLA2* determine arterial vasoconstriction. Arterioscler Thromb Vasc Biol 32:1066-7
Yang, Bo; Gwozdz, Tomasz; Dutko-Gwozdz, Joanna et al. (2012) Orai1 and Ca2+-independent phospholipase A2 are required for store-operated Icat-SOC current, Ca2+ entry, and proliferation of primary vascular smooth muscle cells. Am J Physiol Cell Physiol 302:C748-56
Gwozdz, Tomasz; Dutko-Gwozdz, Joanna; Schafer, Claudia et al. (2012) Overexpression of Orai1 and STIM1 proteins alters regulation of store-operated Ca2+ entry by endogenous mediators. J Biol Chem 287:22865-72
Csutora, Peter; Peter, Krisztina; Kilic, Helena et al. (2008) Novel role for STIM1 as a trigger for calcium influx factor production. J Biol Chem 283:14524-31
Gwozdz, Tomasz; Dutko-Gwozdz, Joanna; Zarayskiy, Vladislav et al. (2008) How strict is the correlation between STIM1 and Orai1 expression, puncta formation, and ICRAC activation? Am J Physiol Cell Physiol 295:C1133-40
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Smani, Tarik; Patel, Tina; Bolotina, Victoria M (2008) Complex regulation of store-operated Ca2+ entry pathway by PKC-epsilon in vascular SMCs. Am J Physiol Cell Physiol 294:C1499-508
Zarayskiy, Vladislav; Monje, Francisco; Peter, Krisztina et al. (2007) Store-operated Orai1 and IP3 receptor-operated TRPC1 channel. Channels (Austin) 1:246-52
Csutora, Peter; Zarayskiy, Vladislav; Peter, Krisztina et al. (2006) Activation mechanism for CRAC current and store-operated Ca2+ entry: calcium influx factor and Ca2+-independent phospholipase A2beta-mediated pathway. J Biol Chem 281:34926-35
Zakharov, Sergey I; Smani, Tarik; Dobrydneva, Yuliya et al. (2004) Diethylstilbestrol is a potent inhibitor of store-operated channels and capacitative Ca(2+) influx. Mol Pharmacol 66:702-7

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