Cardiac Calcium Atpase Dimerization Measured by Fluorescence Resonance Energy Transfer and Chemical Cross-Linking
Date of Award
Doctor of Philosophy (PhD)
The cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA) establishes the intracellular calcium gradient across the sarcoplasmic reticulum membrane. It has been proposed that SERCA forms homo-oligomers that increase the catalytic rate of calcium transport. We investigated SERCA oligomerization in rabbit left ventricular myocytes using a photoactivatable cross-linker. Western blotting of cross-linked SERCA revealed higher molecular weight species consistent with SERCA oligomerization. Fluorescence resonance energy transfer (FRET) measurements in cells transiently transfected with fluorescently-labeled SERCA2a revealed that SERCA readily forms homo-dimers. These dimers formed in the absence or presence of the SERCA regulatory partner, phospholamban (PLB) and were unaltered by PLB phosphorylation or changes in calcium or ATP. Fluorescence lifetime data are compatible with a model in which PLB interacts with a SERCA homo-dimer in a stoichiometry of 1:2. Functional data show that homo-dimerization of SERCA is an important component of SERCA regulation. Together, these results suggest that SERCA forms constitutive homo-dimers in live cells that and dimer formation is not modulated by SERCA conformational poise, PLB binding, or PLB phosphorylation.
Blackwell, Daniel, "Cardiac Calcium Atpase Dimerization Measured by Fluorescence Resonance Energy Transfer and Chemical Cross-Linking" (2016). Dissertations. 2120.
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Copyright © 2016 Daniel Blackwell