Abstract 17293: Dynamic Structure Changes of SERCA in Live Cells Revealed by Fluorescence Microscopy of 2-Color SERCA Constructs
Background: Optimal calcium handling is important for normal cardiac function, and the sarcoendoplasmic calcium ATPase (SERCA) plays a critical role in calcium uptake during the diastole. X-ray crystallography suggests that the SERCA pump transitions between several conformational substates during its catalytic cycle.
Objective: The objective of our present study was to quantify the dynamic conformational changes of SERCA using genetically encoded fluorophores fused to different domains of the SERCA pump.
Methods and results: We engineered a ”2-color SERCA” construct with an N-terminal mCerulean fusion and intrasequence YFP tag. SERCA structure transitions were detected as changes in fluorescence resonance energy transfer (FRET) between the two fluorescent protein tags, as quantified by the 3-cube method, acceptor photobleaching, and fluorescence lifetime imaging microscopy (FLIM). We found that 2-color SERCA FRET decreased from 28% to 13% with addition of SERCA inhibitor thapsigargin (TG) (EC50= 44 nM). We found that there was no difference in the TG-dependent FRET change when 2-color SERCA was coexpressed with the regulatory partner phospholamban (PLB). The data suggest that binding of TG and PLB are not mutually exclusive, and the regulatory complex conformation is not grossly altered by TG. Comparison of 2-color SERCA constructs with the YFP probe fused to different sites provided information about the relative motions of the N-, A-, and P-domains. The data suggest complex contrary movements of SERCA domains during catalytic cycling. We also quantified structure changes as FRET fluctuations of 2-color SERCA using fluorescence correlation spectroscopy (FCS). Large FRET fluctuations indicated structure transitions occuring on a timescale of ∼100 microseconds. The rates of these structure fluctuations increased with calcium and decreased with TG.
Conclusion: Dynamic structure transitions are important features of SERCA catalytic function. We find that 2-color SERCA constructs report these conformational changes, providing new information about the magnitude, direction, and kinetics of SERCA motions. 2-color SERCA constructs may also be useful for screening candidate compounds for modulation of SERCA pump structure and function.
- © 2010 by American Heart Association, Inc.