Mutations in Phospholamban Alter the Structure and Function of the Calcium ATPase Regulatory Complex

Phospholamban (PLB) is an integral membrane protein that plays an important role in regulation of cardiac calcium handling and contractility. PLB exists as a homopentamer in the membrane, which upon deoligomerization into active monomers reversibly inhibits sarco/endoplasmic reticulum calcium ATPase (SERCA). Mutations in PLB that change the PLB monomer-pentamer equilibrium result in dysregulation of SERCA. To determine the structural and regulatory role of the C-terminal residues of PLB in the membranes of living cells, we fused fluorescent protein tags to PLB and SERCA. We then studied the effect of C-terminal alanine substitutions and truncation mutations on PLB oligomerization and SERCA regulation by fluorescence resonance energy transfer (FRET) measurements in live cells. In addition, we also studied the structural and functional consequences of two naturally-occurring missense mutations of PLB that cause heart failure including L39stop (L39X) and Arg9Cys (R9C). Alanine substitution of PLB C-terminal residues significantly altered FRET from PLB to PLB and SERCA to PLB, suggesting a change in quaternary conformation of PLB pentamer and SERCA-PLB regulatory complex. We also quantified a decrease in PLB oligomerization affinity, and an increase in SERCA-PLB binding affinity for alanine mutants. Notably, truncation of only a few C-terminal residues resulted in significant loss of PLB membrane anchoring and mislocalization to the cytoplasm and nucleus. C-terminal truncations including L39X resulted in progressive loss of PLB-PLB FRET, due to a decrease in the apparent affinity of PLB oligomerization. In addition, we quantified a decrease in the binding affinity of truncated PLB including L39X for SERCA, suggesting a change in quaternary conformation of the SERCA-PLB regulatory complex. Furthermore, FRET measurements revealed that R9C-PLB exhibited an increased propensity for oligomerization, and this was further increased by oxidative stress. The R9C also decreased PLB binding to SERCA, and altered the structure of the PLB-SERCA regulatory complex. In addition, we observed that acute expression of R9C-PLB exerts a positively inotropic and lusitropic effect in cardiomyocytes, in contrast to studies of chronic R9C-PLB expression in transgenic mice. Importantly, R9C-PLB exhibited blunted sensitivity to frequency potentiation and β-adrenergic stimulation, two major physiological mechanisms for the regulation of cardiac performance. We conclude that PLB C-terminal residues are critical for localization, oligomerization, and regulatory function. In particular, the PLB C-terminus is an important determinant of the quaternary structure of the SERCA-PLB regulatory complex. Furthermore, the heart failure mutants of PLB including L39X and R9C decrease SERCA inhibition by altering the structure and function of the SERCA-PLB regulatory complex