Super-Inhibitory Phospholamban Mutants Competitively Displace Wild-Type Phospholamban from the Calcium-Free Cardiac Calcium Pump

Abstract

Phospholamban (PLB) regulates the activity of the cardiac Ca pump (SERCA2a). The molecular mechanism by which PLB inhibits SERCA2a activity involves physical binding of PLB to the Ca-free, E2 conformation of the enzyme, specifically, formation of E2•PLB, which prevents Ca binding to SERCA2a. To test how different PLB mutants competitively interact with E2, we examined the ability of a gain-of-function, cross-linkable PLB mutant, N27A,N30C,L37A-PLB (PLB3) to inhibit Ca-ATPase activity and to cross-link to the PLB-tethered pump (SER-20G-PLB) after co-expressing the two proteins in insect cells. SER-20G-PLB, a chimeric WT-SERCA2a-WT-PLB construct, retains a fully catalytic active Ca-pump and an intrinsically regulatory, flexibly anchored PLB-tether. SER-20G-PLB exhibited a rightward-shifted Ca-dependent ATPase activity curve (KCa=0.26 ± 0.03 μM), compared to that of WT-SERCA2a expressed alone (KCa=0.15 ± 0.02 μM). Co-expressed PLB3 super-inhibited the WT-PLB-tethered enzyme, further shifting the Ca-dependent ATPase activity curve to the right (KCa=0.66 ± 0.08 μM). Further, in the Ca-free condition, PLB3 cross-linked to K328 of SER-20G-PLB at the cytoplasmic extension of M4. Importantly, the cross-linking was completely inhibited by micromolar Ca and the SERCA inhibitor tharpsigargin. The Ki values for Ca-dependent inhibition of PLB3 cross-linking to the tethered pump was 0.9 ± 0.1 μM, similar to that obtained using samples with co-expression of PLB3 and WT-SERCA2a. A similar gain-of-function mutant N27A,L37A,V49C-PLB, also super-inhibited the tethered pump, and cross-linked to V89C-SER-20G-PLB at the C-terminal end of transmembrane domain M2. These results demonstrate that freely diffusing, gain-of-function PLB mutants completely replace the WT-PLB-tether and must fit into the binding pocket previously occupied by WT-PLB. Thus, there is a reversible equilibrium between different PLB mutants and WT-PLB for binding to E2, in which PLB mutants possessing higher binding affinity for SERCA2a produce more stable E2•PLB and lower Ca affinity.