Probing the Structural Mechanism of Cardiac Calcium Pump Regulation by Phospholamban with Site-Directed Spin Labeling

Abstract

Proper calcium transport is critical to muscle function, and the calcium pump sarcoplasmic reticulum Ca-ATPase (SERCA) plays a key role in facilitating muscle relaxation by sequestering calcium ions in muscle cells. Reduced SERCA activity is associated with heart failure, making SERCA an important subject of study for seeking heart failure treatment. The small membrane protein phospholamban (PLB) inhibits cardiac SERCA activity, and phosphorylation of PLB substantially restores SERCA activity, but the structural mechanism behind this process is not well understood. Previous spectroscopic studies on the SERCA-PLB complex have focused on the cytoplasmic domain of PLB, showing a structural shift of the cytoplasmic domain relative to SERCA that accompanies phosphorylation of PLB at Ser-16. Since it has been shown that only expression of PLB's transmembrane domain is necessary for observing SERCA inhibition, in this work, we performed site-directed spin labeling and EPR accessibility measurements on the transmembrane domain. We show that SERCA binding reduces access of the transmembrane domain to the lipid environment, and phosphorylation of PLB alters its topology without dissociation from SERCA.