Author

Gauri Akolkar

Date of Award

4-2011

Degree Type

Thesis

Degree Name

Master of Science (MSc)

Department

Biology

Supervisor

A.K. Grover

Language

English

Abstract

Calcium (Ca2+) is an important signaling molecule with tightly regulated movements across cell membranes. Ca2+ transporters play a key role in this regulation. The sarco/endoplasmic reticulum (SER) Ca2+ pump (SERCA) and plasma membrane Ca2+ pump (PMCA) keep cytosolic Ca2+ levels [Ca2+]i low. The sodium calcium exchanger (NCX) is bidirectional and may work to expel Ca2+ or bring it into the cell depending on the electrochemical gradient. NCX has been proposed to play a role in refilling the SER Ca2+ pool.

The overall objective of this thesis was to determine the effect of SER Ca2+ depletion on NCX- SERCA interactions in pig coronary artery smooth muscle by monitoring changes in [Ca2+]i and the co-localization between NCX and SERCA. NCX mediated increase in [Ca2+]i was observed in Na+ loaded smooth muscle cells. The effect of SER depletion by thapsigargin was examined on the NCX mediated increase in [Ca2+]i. A decrease in the NCX mediated increase in [Ca2+]i was observed upon inhibition of SERCA pump with thapsigargin. The effect of SER depletion on proximity of NCX and SERCA proteins was also investigated using immunofluorescence confocal microscopy using anti-NCXl and anti-SERCA2 antibodies and fluorescence labeled secondary antibodies. Na+ loaded smooth muscle cells in NMG buffer were incubated with or without SERCA inhibitor thapsigargin. Resulting image stacks were analyzed for the co-localization of NCXl and SERCA2 in the areas near the plasma membrane. SER depletion with thapsigargin increased the co-localization between NCXl and SERCA2 near the plasma membrane of smooth muscle cells. Thus, SER Ca2+ depletion moves plasma membrane protein NCX and SER protein SERCA closer to each other and decreases the NCX mediated Ca2+ entry in the cell. These interactions may be crucial to smooth muscle Ca2+ regulation and has the ability to be a potential therapeutic target during oxidative stress. However, the nature of these interactions needs to be explored.

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