We have used functional co-reconstitution of purified sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) with phospholamban (PLB), its inhibitor in the heart, to test the hypothesis that loss-of-function (LOF) PLB mutants (PLBM) can compete with wild-type PLB (PLBW) to relieve SERCA inhibition. Co-reconstitution at varying PLB-to-SERCA ratios was conducted using synthetic PLBW, 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 Ca2+-dependence of ATPase activity. At saturating PLB, the inhibitory potency of I40A was about three times that of PLBW, while the potency of each of the LOF PLBM was about one third that of PLBW. However, there was no significant variation in the apparent SERCA affinity for these four PLB variants. When SERCA was co-reconstituted with mixtures of PLBW and LOF PLBM, inhibitory potency was reduced relative to that of PLBW alone. Furthermore, FRET between donor-labeled SERCA and acceptor-labeled PLBW was decreased by both (unlabeled) LOF PLBM. These results show that LOF PLBM can compete both physically and functionally with PLBW, 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 PLBM targeted for gene therapy of heart failure in humans. (C) 2011 Elsevier Inc. All rights reserved.