Ca2+ Signaling in the Near Plasma Membrane Microdomain of Non-Excitable Cells
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.