Abstract
To investigate the mechanism of regulation of sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) by phospholamban (PLB), we expressed Cerulean-SERCA and yellow fluorescent protein (YFP)-PLB in adult rabbit ventricular myocytes using adenovirus vectors. SERCA and PLB were localized in the sarcoplasmic reticulum and were mobile over multiple sarcomeres on a timescale of tens of seconds. We also observed robust fluorescence resonance energy transfer (FRET) from Cerulean-SERCA to YFP-PLB. Electrical pacing of cardiac myocytes elicited cytoplasmic Ca(2+) elevations, but these increases in Ca(2+) produced only modest changes in SERCA-PLB FRET. The data suggest that the regulatory complex is not disrupted by elevations of cytosolic calcium during cardiac contraction (systole). This conclusion was also supported by parallel experiments in heterologous cells, which showed that FRET was reduced but not abolished by calcium. Thapsigargin also elicited a small decrease in PLB-SERCA binding affinity. We propose that PLB is not displaced from SERCA by high calcium during systole, and relief of functional inhibition does not require dissociation of the regulatory complex. The observed modest reduction in the affinity of the PLB-SERCA complex with Ca(2+) or thapsigargin suggests that the binding interface is altered by SERCA conformational changes. The results are consistent with multiple modes of PLB binding or alternative binding sites.
Highlights
Quantification of fluorescence resonance energy transfer (FRET) from sarco-endoplasmic reticulum Ca2؉-ATPase (SERCA) to PLB in Cardiac Myocytes—Confocal imaging of Cer-SERCA expressed in adult rabbit ventricular myocytes showed a striated pattern of fluorescence as well as longitudinal streaks (Fig. 2A), suggesting localization in the junctional and transverse sarcoplasmic reticulum, respectively
FRET between Cer-SERCA and yellow fluorescent protein (YFP)-PLB was quantified in these myocytes with the E-FRET method [17, 29]
We investigated the effect of SERCA inhibitor Tg on SERCA-PLB FRET because this ligand is reported to abolish cross-linking of the regulatory complex [10, 30, 31]
Summary
Cross-linking was prevented by the SERCA inhibitor thapsigargin (Tg) (7, 9 –11) Another recent study showed that oligomerization of a PLB-SERCA fusion construct was increased by micromolar Ca2ϩ [13], consistent with the idea that Ca2ϩ causes displacement of PLB from the inhibitory cleft, permitting PLB self-association into pentamers. Mueller et al [14] showed that FRET was decreased, but not abolished, by Ca2ϩ This result suggested that relief of inhibition was accomplished by a conformational change of the regulatory complex, rather than unbinding of PLB from the pump. The investigators suggested that the regulator PLB may be considered a subunit of the pump and remains bound to SERCA throughout the catalytic cycle (Fig. 1B, Subunit Model) To test these alternative mechanisms, we expressed SERCA and PLB fused to fluorescent proteins to quantify the regulatory interaction by FRET in live cells. We expressed fluorescently labeled SERCA and PLB in adult ventricular myocytes to determine whether the regulatory complex is dynamically dissociated during the cardiac cycle
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
