Electric Studies of Excitation, Secretion & Contraction

University Of Washington, Seattle WA

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Abstract

DESCRIPTION (provided by applicant): Calcium signaling and the regulation of exocytosis are central issues in the physiology of all animal cells. This grant seeks quantitative understanding of such signaling through biophysical experiments in electrically excitable and non-excitable mammalian cell lines: PC12 pheochromocytoma cells, tsA epithelial cells, and pancreatic duct epithelial cells. A few experiments will use rodent primary chromaffin cells. Two long-term hypotheses guide this work: That Ca2+ clearance and the regulation of exocytosis take different forms in different cells and are tuned to the physiological role of each cell;and that several intracellular organelles make significant contributions to cellular Ca2+ dynamics. The aims in this grant period are: (1) To test the hypothesis that accumulation and release of Ca2+ by secretory granules can make significant contributions to cellular Ca2+ signaling during physiological responses. (2) To measure the amplitude of receptor evoked inositol 1,4,5, trisphosphate (IP3) elevations and to test the hypothesis that Ca2+ signaling via IP3 is terminated by rapid metabolism of IP3 by IP3 5- phosphatase followed by rapid reuptake of Ca2+ into the endoplasmic reticulum Ca2+ stores. And (3) To test the hypothesis that cytoskeletal tracks and fast cytoskeletal remodeling participate in the mobilization of secretory granules from reserve pools into secretion-competent pools. The work requires a range of biophysical techniques including: patch clamp of ion currents;amperometric and capacitance measurements of exocytosis;transfection of genetically targeted probes, indicators, and cellular proteins;ratiometric photometry and fluorescence resonance energy transfer (FRET) of indicators;video fluorescence imaging;total internal reflection microscopy (TIRF);confocal microscopy;quantitative kinetic modeling.