Project Summary Spatially and temporally precise Ca2+ signals control a vast spectrum of cellular functions including contraction, secretion, transcription, and growth. These signals are mediated by the coordinated function of Ca2+ sensors and Ca2+ channels. STIM proteins in the endoplasmic reticulum (ER) are luminal Ca2+ sensors in the endoplasmic reticulum (ER) that physically couple to highly Ca2+-selective plasma membrane Orai channels, through an extraordinarily dynamic inter-membrane ER-PM junctional coupling process, to generate "store-operated" Ca2+ signals. Dysregulation of STIM/Orai expression is associated with a range of disorders including renal fibrosis, Idiopathic pulmonary fibrosis, immune deficiency, muscle weakness, skin dysplasia, and cancer. The proposed studies address the crucial unsolved questions of how STIM proteins undergo activation, what is the molecular basis of coupling between the distinct STIM and Orai isoforms to generate Ca2+ signals, and what is the pathophysiological role that the STIM/Orai pathway in cellular remodeling in disease, in particular, in the mediation of fibrosis, a major feature in multiple chronic diseases leading to global morbidity. Understanding this fundamental signaling mechanism provides critical new information to generate pharmacological tools to modify Ca2+ signals and alleviate diseases of cellular growth and remodeling. Genetic alterations giving rise to gain or loss of function of STIM and Orai proteins lead to severe immunological, muscular, skin, and neural deficits in humans, and provide critical molecular insights into the structure/function of STIM/Orai proteins. To deepen our understanding of this critical signaling pathway, the project goals focus on: (1) Investigating the mechanisms underlying STIM protein activation by Ca2+ store-sensing or temperature change, and the precise molecular coupling of STIM proteins to Orai channels, focusing on the highly distinct STIM and Orai isoforms, and utilizing precise molecular/genetic probes, imaging technology, and gene-deleted cell lines and animal models: (2) Studying how the small molecule 2-aminoethyldiphenyl borate (2-APB) specifically modulates STIM/Orai isoforms, using genetically-encoded and optogenetically/chemogenetically applied Ca2+ probes tagged onto STIM and Orai proteins to monitor cytosolic Ca2+ signals mediated by STIM/Orai, and exploring how 2-APB provides crucial information on STIM-mediated clustering of Orai channels and controls local junctional Ca2+ signals, advancing our mechanistic understanding and the development of small molecules targeting the STIM/Orai pathway; (3) Examining how STIM/Orai-mediated calcium signals regulate fibrosis in both cellular and animal model systems, focusing on how the STIM1/Orai2 isoform pathway can be modified to regulate the process of fibrosis, determining transcriptomal changes related to STIM-specific Ca2+ signal generation, and utilizing a novel STIM-specific inhibitory peptid...