Store-operated Ca2+ entry (SOCE) in skeletal muscle is mediated by coupling between Stim1 Ca2+ sensors in the sarcoplasmic reticulum (SR) and Ca2+-permeable Orai1 channels in the transverse tubule (TT). SOCE activity is also modulated by calsequestrin-1 (Casq1), the primary SR Ca2+ buffer in muscle. SOCE enhances muscle development, limits fatigue, and promotes fatigue-resistant type I fiber specification. On the other hand, SOCE dysfunction contributes to muscle weakness in aging, exacerbates muscular dystrophy, and mutations in Stim1, Orai1, and Casq1 all result in a myopathy characterized by the presence of tubular aggregates. Thus, Orai1-dependent SOCE activity plays a critical role in both normal muscle function and disease. We recently found that acute exercise drives the formation of new SR-TT junctions (Ca2+ entry units, CEUs). CEUs promote Orai1-dependent constitutive and store-operated Ca2+ entry that enhances SR store refilling, Ca2+ release, and force production during repetitive stimulation. Interestingly, CEUs are also constitutively present in sedentary mice that lack Casq1 and wild type mice after 1 month of voluntary wheel running (VWR). For this project, we developed knock-in mice with a V5-3xHA epitope tag on the extreme Orai1 C-terminus (Orai1V5HA/+ mice). Using these mice, we identified two distinct Orai1 isoforms (short and long) in skeletal muscle (both being heavily glycosylated), but only one isoform in spleen. Our preliminary data demonstrate the feasibility of using Orai1V5HA/+ mice to identify the Orai1 interacting proteins in muscle at rest and after acute exercise. Parallel studies will be conducted using N-terminal and C-terminal Orai1-TurboBioID tandem constructs following introduction in muscle of inducible, muscle-specific Orai1 knockout mice. The proposed “high risk/high reward” studies will employ cutting-edge, non-biased proteomic approaches (HA-IP, proximity biotinylation) to identify changes in Orai1 interacting proteins (“Orai1 interactome”) in skeletal muscle under conditions that promote CEU formation and SOCE activity (acute exercise, VWR, and Casq1 deficiency). We will use these discoveries, research tools, and cutting-edge approaches to determine the molecular mechanisms that coordinate Orai1-dependent CEU formation, stabilization and disassembly. We hypothesize that: 1) acute exercise drives Orai1 protein interactions that trigger a macromolecular motor to drive dynamic and reversible CEU formation and 2) challenges to SR Ca2+ store content (prolonged VWR, Casq1 deficiency) promote Orai1 protein interactions that stabilize CEUs. Aim 1 will interrogate the molecular mechanism that coordinates dynamic, exercise-dependent CEU formation by characterizing the Orai1 interactome in muscle at rest and after acute exercise. Aim 2 will characterize the Orai1 interactome in muscle under conditions that favor the stable formation of CEUs (long-term VWR and Casq1 deficiency). These studies will delineate the molec...