Functional and physical competition between phospholamban and its mutants provides insight into the molecular mechanism of gene therapy for heart failure.

We have used functional co-reconstitution of purified sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA) with phospholamban (PLB), its inhibitor in the heart, to test the hypothesis that loss-of-function (LOF) PLB mutants (PLB(M)) can compete with wild-type PLB (PLB(W)) to relieve SERCA inhibition. Co-reconstitution at varying PLB-to-SERCA ratios was conducted using synthetic PLB(W), gain-of-function mutant I40A, or LOF mutants S16E (phosphorylation mimic) or L31A. Inhibitory potency was defined as the fractional increase in K(Ca), measured from the Ca(2+)-dependence of ATPase activity. At saturating PLB, the inhibitory potency of I40A was about three times that of PLB(W), while the potency of each of the LOF PLB(M) was about one third that of PLB(W). However, there was no significant variation in the apparent SERCA affinity for these four PLB variants. When SERCA was co-reconstituted with mixtures of PLB(W) and LOF PLB(M), inhibitory potency was reduced relative to that of PLB(W) alone. Furthermore, FRET between donor-labeled SERCA and acceptor-labeled PLB(W) was decreased by both (unlabeled) LOF PLB(M). These results show that LOF PLB(M) can compete both physically and functionally with PLB(W), provide a rational explanation for the partial success of S16E-based gene therapy in animal models of heart failure, and establish a powerful platform for designing and testing more effective PLB(M) targeted for gene therapy of heart failure in humans.