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 i n 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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM083913-36
Application #
8053258
Study Section
Neurotransporters, Receptors, and Calcium Signaling Study Section (NTRC)
Program Officer
Gindhart, Joseph G
Project Start
1977-09-01
Project End
2012-03-31
Budget Start
2011-04-01
Budget End
2012-03-31
Support Year
36
Fiscal Year
2011
Total Cost
$336,370
Indirect Cost
Name
University of Washington
Department
Physiology
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Jung, Seung-Ryoung; Deng, Yi; Kushmerick, Christopher et al. (2018) Minimizing ATP depletion by oxygen scavengers for single-molecule fluorescence imaging in live cells. Proc Natl Acad Sci U S A 115:E5706-E5715
Jung, Seung-Ryoung; Seo, Jong Bae; Deng, Yi et al. (2016) Contributions of protein kinases and ?-arrestin to termination of protease-activated receptor 2 signaling. J Gen Physiol 147:255-71
Yu, Haijie; Dickson, Eamonn J; Jung, Seung-Ryoung et al. (2016) High membrane permeability for melatonin. J Gen Physiol 147:63-76
Yu, Haijie; Benitez, Sergio G; Jung, Seung-Ryoung et al. (2016) GABAergic signaling in the rat pineal gland. J Pineal Res 61:69-81
Yu, Haijie; Seo, Jong Bae; Jung, Seung-Ryoung et al. (2015) Noradrenaline upregulates T-type calcium channels in rat pinealocytes. J Physiol 593:887-904
Seo, Jong Bae; Moody, Mark; Koh, Duk-Su (2014) Epithelial monolayer culture system for real-time single-cell analyses. Physiol Rep 2:e12002
Liang, Kuo; Du, Wen; Lu, Jingze et al. (2014) Alterations of the Ca²? signaling pathway in pancreatic beta-cells isolated from db/db mice. Protein Cell 5:783-94
Yoon, Jin-Young; Jung, Seung-Ryoung; Hille, Bertil et al. (2014) Modulation of nicotinic receptor channels by adrenergic stimulation in rat pinealocytes. Am J Physiol Cell Physiol 306:C726-35
Falkenburger, Björn H; Dickson, Eamonn J; Hille, Bertil (2013) Quantitative properties and receptor reserve of the DAG and PKC branch of G(q)-coupled receptor signaling. J Gen Physiol 141:537-55
Kim, Mean-Hwan; Seo, Jong Bae; Burnett, Lindsey A et al. (2013) Characterization of store-operated Ca2+ channels in pancreatic duct epithelia. Cell Calcium 54:266-75

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