Cytoplasmic interactions between phospholamban residues 1-20 and the calcium-activated ATPase of the sarcoplasmic reticulum

Phospholamban regulates the activity of the calcium-activated ATPase (CaATP ase) of cardiac sarcoplasmic reticulum. Equilibrium fluorescence studies ha ve shown that the N-terminal cytoplasmic region of phospholamban (residues 1-20, domain 1) causes a decrease in the intrinsic tryptophan fluorescence of the CaATPase. The interaction of phospholamban residues 1-20 with the Ca ATPase also results in spectral changes for the extrinsic chromophore FITC covalently attached to the cytoplasmic region of the calcium pump. The fluo rescence changes for both reporter groups correlate with a dissociation con stant of approximate to 40 muM for the complex between phospholamban residu es 1-20 and the CaATPase. Complex formation is notably weaker when phosphol amban 1-20 is titrated into the CaATPase in the presence of calcirun, with altered conformational effects resulting from binding. The interaction of d omain 1 of phospholamban with the CaATPase is also reduced upon phosphoryla tion of phospholamban 1-20 at Ser-16. This region of phospholamban 1-20 is shown by isotope-edited NMR study to be involved in interaction with the Ca ATPase. Binding of the phosphorylated peptide is not abolished, however, in dicating that phospholamban 1-20 remains associated with the CaATPase even after phosphorylation.The data provide direct evidence for the interaction between the cytoplasmic regions of phospholamban and the pump, and are disc ussed in the context of the mechanism for inhibition of cardiac CaATPase ac tivity by phospholamban.