The long term goal of this proposal is to understand the role of intracellular calcium ([Ca2+]i) signals in the function of cytotoxic T lymphocytes (CTLs). These critical effectors of the immune system kill virally infected cells and cancer cells, and play a major role in the immune response to transplanted tissues; inappropriate killing can cause autoimmune diseases such as Lupus, certain forms of diabetes, and arthritis. CTL function is therefore of critical relevance to health and disease, and detailed knowledge of how CTLs kill is of key importance to understanding the etiology of viral diseases such as AIDS, cancer autoimmune disorders, and to preventing rejectio of transplanted organs. Therapeutic strategies may be suggested by detailed knowledge of CTL function. One of the two mechanisms CTLs use to kill is the perforin pathway, which involves the exocytotic release of pore-forming peptides and hydrolytic enzyme contained in specialized CTL lytic granules into an area of close apposition formed with the target. Granule exocytosis is a two step process, involving relocation of lytic granules followed by their fusion, and is known to require increased [Ca2+]i caused by influx across the plasma membrane. However, the specific role(s) of Ca2+ in driving granule exocytosis is unknown. Critically, granule exocytosis has not been studied using the powerful physiological approaches that have advanced our understanding of exocytosis in other cell types.
The specific aims of this proposal are to use patch clamp recording, capacitance measurements and digital video imaging techniques to: 1) determine the mechanism of Ca2+ influx in CTLs; 2) investigate the idea that Ca2+ channels are localized to create functional Ca2+ gradients; and 3) determine the role(s) played by Ca2+ in granule exocytosis.
These specific aims will establish a firm physiological foundation for understanding the mechanism of this critical exocytotic event.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI042964-04
Application #
6373821
Study Section
Physiology Study Section (PHY)
Program Officer
Quill, Helen R
Project Start
1998-04-01
Project End
2003-03-31
Budget Start
2001-04-01
Budget End
2002-03-31
Support Year
4
Fiscal Year
2001
Total Cost
$155,891
Indirect Cost
Name
University of Colorado Denver
Department
Physiology
Type
Schools of Medicine
DUNS #
065391526
City
Aurora
State
CO
Country
United States
Zip Code
80045
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Zweifach, A (2000) Target-cell contact activates a highly selective capacitative calcium entry pathway in cytotoxic T lymphocytes. J Cell Biol 148:603-14
Zweifach, A (2000) FM1-43 reports plasma membrane phospholipid scrambling in T-lymphocytes. Biochem J 349:255-60