Probing cardiac calcium regulation using time-resolved optical spectroscopy.

Li, Ji
2012-07
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Probing cardiac calcium regulation using time-resolved optical spectroscopy.

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2012-07

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Sarcoplasmic reticulum calcium adenosine triphosphatase (SERCA) is an integral membrane Ca2+ pump that reuptakes Ca2+ into the lumen of sarcoplasmic reticulum (SR), decreases the cytoplasmic Ca2+ concentration, and initiates relaxation in both skeletal and cardiac muscle. In the heart, SERCA activity is partially inhibited by phospholamban, another integral membrane protein. This inhibition can be relieved by high Ca2+ concentration or phosphorylation of PLB. Misregulation of Ca2+ by the SERCA-PLB complex is associated with heart failure. The goal of this thesis is to investigate the mechanism of Ca2+ regulation by the SERCA-PLB complex, by direct measurement of their physical interactions using optical spectroscopy. We have used time-resolved fluorescence resonance energy transfer (TRFRET) to determine the binding and distance distribution between the donor-labeled SERCA and acceptor-labeled SERCA-bound PLB in artificial membranes. Results show that PLB binds to SERCA with two structures, an extended R state with the cytoplasmic domain pointing away from the membrane surface, resulting in a shorter interprobe distance, and a T state with the cytoplasmic domain bound to the membrane, giving rise to a longer interprobe distance. We modulated the electrostatic interaction between SERCA and PLB by adjusting the membrane headgroup charge. Results show that the T state of PLB is more inhibitory than R. SERCA inhibition by PLB is well correlated with the T/R equilibration and does not require dissociation of the complex. We used time-resolved phosphorescence anisotropy (TPA) to analyze the functional oligomeric regulation of SERCA by Ca2+ and PLB phosphorylation in native cardiac SR. A uniaxial rotation diffusion analysis of TPA data was used to resolve the oligomeric state of SERCA. We found that SERCA, in both the skeletal and cardiac SR, consists of 3 oligomeric species plus an immobilized large aggregate. SERCA activation by either Ca2+ or PLB phosphorylation correlates with the destabilization of the immobilized large aggregate, without significantly affecting the sizes of the 3 oligomeric states. In summary, PLB regulates SERCA through two independent mechanisms. In the bound SERCA-PLB complex, the structural dynamics of PLB determines its inhibitory effect on SERCA.. In addition, Ca2+ and phosphorylation of PLB regulate SERCA activity by dissociating the immobilized SERCA aggregates and increasing the functional oligomeric states.

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University of Minnesota Ph.D. dissertation. July 2012. Major: Physics. Advisor:David D. Thomas. 1 computer file (PDF); xiii, 104 pages.

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Li, Ji. (2012). Probing cardiac calcium regulation using time-resolved optical spectroscopy.. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/133862.

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