The project aims to understand how unique signatures of Ca2+ mediated by STIM proteins control the proliferation of vascular smooth muscle cells (VSMC). Ca2+ signals play a crucial role in not only controlling vascular contraction but also in regulating the growth and proliferation of smooth muscle cells. The work examines the function of two crucial proteins, STIM1 and STIM2, that sense changes in the Ca2+ within the SR lumen of VSMCS, and through a highly coordinated translocation process, move into small specialized junctions between the SR and PM. STIM proteins directly activate Ca2+ channels in the PM and hence control Ca2+ entry into VSMCS. The project has two specific aims: Aim 1: To examine the distinct functional roles of STIM1 and STIM2 proteins in mediating Ca2+ signals in VSMCs. These studies will test the hypothesis that the STIM2 phenotype in VSMCs from SM-STIM1-KO animals has a "Ca2+ signature response" important in mediating distinct physiological differences in VSMC proliferation. The experimental approach to test this hypothesis utilizes a combination of live cellular imaging, expression of dominant negative channel proteins, electrophysiology, and novel Ca2+ probes to assess the functional roles of STIM-mediated Ca2+ signals in VSMCs and examines: (a) the "temporal Ca2+ signature" of Ca2+ signals in VSMCs from SM-STIM1- KO mice;(b) the "spatial Ca2+ signature" of Ca2+ signals in VSMCs from SM-STIM1-KO mice;and (c) how luminal SR levels reflect the STIM-mediated Ca2+ signature in VSMCs. Aim 2: To determine how STIM-induced Ca2+ signature responses regulate proliferative pathways in VSMCs. The hypothesis to be tested is that STIM-specific Ca2+ entry signals control gene expression and proliferative VSMC responses through the calcineurin/NFAT axis. The aims are to determine (a) how STIM proteins control expression of components of the calcineurin NFAT pathway in proliferative VSMC;(b) STIM-mediated Ca2+ signaling occurs during mitogenic stimulation;(c) how STIM proteins control the NFAT translocation/gene expression pathway.