Oligomeric Interactions in Cardiac Calcium Regulation

Abstract

We used time-resolved fluorescence resonance energy transfer (TR-FRET) to determine the oligomeric state of phospholamban (PLB) and its inhibited target, sarcoplasmic reticulum Ca-ATPase (SERCA). Previous work on our lab has shown that PLB is primarily pentameric but SERCA binds preferentially to the monomeric form in lipid vesicles. Recent EM studies suggest that the PLB pentamer might also bind to SERCA. We tested this hypothesis by labeling SERCA at C674 with a fluorescent donor (TMRIA) and labeling PLB at K3 with a non-fluorescent acceptor (MGITC), then reconstituting the proteins into lipid vesicles and performing TR-FRET as a function of the fraction of acceptor-labeled PLB (xA), keeping the total PLB/SERCA molar ratio constant at 10. Simulations showed that if a PLB monomer binds to SERCA, the dependence of FRET on xA should be linear, but the binding of a PLB oligomer should produce distinct curvature in the plot. The observed plot was quite linear, and was indistinguishable from control experiments using a monomeric mutant of PLB. We conclude that PLB binds to SERCA only as a monomer. These results have important implications for the design of PLB mutants to be used in gene therapy for heart failure.