Characterizing Phospholamban to Sarco(endo) plasmic Reticulum Ca2+-ATPase 2a (SERCA2a) Protein Binding Interactions in Human Cardiac Sarcoplasmic Reticulum Vesicles Using Chemical Cross-linking

Chemical cross-linking was used to study protein binding interactions between native phospholamban (PLB) and SERCA2a in sarcoplasmic reticulum (SR) vesicles prepared from normal and failed human hearts. Lys(27) of PLB was cross-linked to the Ca2+ pump at the cytoplasmic extension of M4(at or near Lys(328)) with the homobifunctional cross-linker, disuccinimidyl glutarate (7.7 angstrom). Cross-linking was augmented by ATP but abolished by Ca2+ or thapsigargin, confirming in native SR vesicles that PLB binds preferentially to E2 (low Ca2+ affinity conformation of the Ca2+-ATPase) stabilized by ATP. To assess the functional effects of PLB binding on SERCA2a activity, the antiPLB antibody, 2D12, was used to disrupt the physical interactions between PLB and SERCA2a in SR vesicles. We observed a tight correlation between 2D12-induced inhibition of PLB cross-linking to SERCA2a and 2D12 stimulation of Ca2+ ATPase activity and Ca2+ transport. The results suggest that the inhibitory effect of PLB on Ca2+-ATPase activity in SR vesicles results from mutually exclusive binding of PLB and Ca2+ to the Ca2+ pump, requiring PLB dissociation for catalytic activation. Importantly, the same result was obtained with SR vesicles prepared from normal and failed human hearts; therefore, we conclude that PLB binding interactions with the Ca2+ pump are largely unchanged in failing myocardium.