Ca2+ Signaling in the Near Plasma Membrane Microdomain of Non-Excitable Cells
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Calcium is the most versatile second messenger and plays fundamental roles in orchestrating enzyme secretion in exocrine acinar cells. Previous studies in excitable cells demonstrated the existence of high Ca2+ microdomains. The major function of such microdomains is to create high local calcium concentration to activate various calcium-dependent signaling events. However, in non-excitable cells, direct evidence of such microdomains is absent. The goal of my study is to characterize the properties of high Ca2+ microdomains in acinar cells and explore its physiological relevance in the context of the secretory functions. By combining Total Internal Reflection Fluorescence Microscopy (TIRFM) technique and wide-field fluorescence imaging, I was able to quantify and compare changes in the concentration of free Ca2+ in the near membrane microdomains (?[Ca2+]PM) and in the bulk cytosol (?[Ca2+]Cyto). ?[Ca2+]PM is about 3-fold larger than ?[Ca2+]Cyto under maximal agonist stimulation, while resting [Ca2+]PM and [Ca2+]Cyto shows no difference. Near membrane microdomains also showed greater Ca2+ influx following store depletion induced either by activating surface receptor or by inhibiting SERCA pump. In response to physiological strength of stimulation, Ca2+ oscillation in the two compartments showed significantly different dynamics. The activation mechanisms of the Ca2+-induced Ca2+ release (CICR) are well established in cardiac and skeletal muscles and involves high Ca2+ microdomains. My study was the first to demonstrate the presence of CICR in the parotid acinar cell. In these cells, minimal activation of Ca2+ influx by partially depleting the stores, either by directly activating the cell surface receptor or by inhibiting SERCA, leads to an explosive release of Ca2+ from the majority of the stores, mediated presumably by RyR away from microdomains. The last part of my study is on the effects of chronic ER stress on Ca2+ signaling. The study suggests that ER stress induced by PERK mutation impeded both the efficiency and fidelity of Ca2+ signaling. My work validates the existence of near plasma membrane microdomains in non-excitable exocrine cells. The fact that [Ca2+]PM and [Ca2+]Cyto differ in many ways suggests that microdomains is the central signaling platform in these cells.