The physical and mechanistic basis for calcium-ATPase regulation by phospholamban.

The Ca-ATPase of sarcoplasmic reticulum is a 110 kDa integral membrane protein responsible for calcium transport from the cytosol into the sarcoplasmic reticulum lumen to promote muscle relaxation. In the heart, the Ca-ATPase is regulated by phospholamban, a 52 residue integral membrane protein, which inhibits the Ca-ATPase by decreasing the apparent calcium affinity of the enzyme. Phospholamban phosphorylation relieves Ca-ATPase inhibition. At present, the physical mechanism by which phospholamban regulates the Ca-ATPase is not known. The purpose of this dissertation research was to test the hypothesis that phospholamban regulates the CaATPase by altering Ca-ATPase protein-protein interactions that are important for high affinity Ca2+ binding to the enzyme. For these studies, the cardiac Ca-ATPase (SERCA2a isoform) was expressed either in the absence (control studies) or presence of coexpressed phospholamban using the baculovirus-High Five insect cell expression system, and isolated as crude microsomal fractions. We first used saturation-transfer electron paramagnetic resonance (ST-EPR) spectroscopy to determine the effect of phospholamban on Ca-ATPase rotational mobility and average oligomeric state. We developed a method to spin label the Ca-ATPase in microsomes with the maleimide spin label, in which 80% of the spin label was bound selectively to the Ca-ATPase. ST-EPR analysis indicated that Ca-ATPase rotational mobility in the absence of phospholamban was indicative of a tetramer. In the presence of coexpressed phospholamban, Ca-ATPase rotational mobility was increased by 15–20%. The results suggest that phospholamban induced a rearrangement of the Ca-ATPase subunits within the tetramer, rather than decreasing the average oligomeric size of the enzyme. Next, rapid mixing methods were used to determine the effects of phospholamban on Ca-ATPase phosphorylation kinetics, which are sensitive to Ca-ATPase protein-protein interactions. Phospholamban induced significant changes in the rates and amplitudes of Ca-ATPase phosphoenzyme formation and decay and altered the steady-state ratio of the ADP-sensitive and ADP-insensitive phosphoenzymes. The kinetics results are consistent with decreased Ca-ATPase protein-protein interactions in the presence of phospholamban. The results of these studies are discussed in terms of the effect of phospholamban on Ca-ATPase calcium binding affinity and calcium transport activity during heart muscle relaxation