Mechanism of reversal of phospholamban inhibition of the cardiac Ca2+-ATPase by protein kinase a and by anti-phospholamban monoclonal antibody 2D12

Our model of phospholamban (PLB) regulation of the cardiac Ca2+ -ATPase in sarcoplasmic reticulum (SERCA2a) states that PLB binds to the Ca2+ -free, E2 conformation of SERCA2a and blocks it from transitioning from E2 to E1, the Ca2+ -bound state. PLB and Ca2+ binding to SERCA2a are mutually exclusive, and PLB inhibition of SERCA2a is manifested as a decreased apparent affinity of SERCA2a for Ca2+. Here we extend this model to explain the reversal of SERCA2a inhibition that occurs after phosphorylation of PLB at Ser(16) by protein kinase A (PKA) and after binding of the anti-PLB monoclonal antibody 2D12, which recognizes residues 7-13 of PLB. Site-specific cysteine variants of PLB were co-expressed with SERCA2a, and the effects of PKA phosphorylation and 2D12 on Ca2+- ATPase activity and cross-linking to SERCA2a were monitored. In Ca2+- ATPase assays, PKA phosphorylation and 2D12 partially and completely reversed SERCA2a inhibition by decreasing K-Ca values for enzyme activation, respectively. In cross-linking assays, cross-linking of PKA-phosphorylated PLB to SERCA2a was inhibited at only two of eight sites when conducted in the absence of Ca2+ favoring E2. However, at a subsaturating Ca2+ concentration supporting some E1, cross-linking of phosphorylated PLB to SERCA2a was attenuated at all eight sites. KCa values for cross-linking inhibition were decreased nearly 2-fold at all sites by PLB phosphorylation, demonstrating that phosphorylated PLB binds more weakly to SERCA2a than dephosphorylated PLB. In parallel assays, 2D12 blocked PLB cross-linking to SERCA2a at all eight sites regardless of Ca2+ concentration. Our results demonstrate that 2D12 restores maximal Ca2+-ATPase activity by physically disrupting the binding interaction between PLB and SERCA2a. Phosphorylation of PLB by PKA weakens the binding interaction between PLB and SERCA2a ( yielding more PLB-free SERCA2a molecules at intermediate Ca2+ concentrations), only partially restoring Ca2+ affinity and Ca2+- ATPase activity.