The sarcoplasmic reticulum calcium pump (SERCA) and its regulator, 
phospholamban, are essential components of cardiac contractility. Phospholamban 
modulates contractility by inhibiting SERCA, and this process is dynamically 
regulated by β-adrenergic stimulation and phosphorylation of phospholamban. 
Herein we reveal mechanistic insight into how four hereditary mutants of 
phospholamban, Arg(9) to Cys, Arg(9) to Leu, Arg(9) to His, and Arg(14) 
deletion, alter regulation of SERCA. Deletion of Arg(14) disrupts the protein 
kinase A recognition motif, which abrogates phospholamban phosphorylation and 
results in constitutive SERCA inhibition. Mutation of Arg(9) causes more complex 
changes in function, where hydrophobic substitutions such as cysteine and 
leucine eliminate both SERCA inhibition and phospholamban phosphorylation, 
whereas an aromatic substitution such as histidine selectively disrupts 
phosphorylation. We demonstrate that the role of Arg(9) in phospholamban 
function is multifaceted: it is important for inhibition of SERCA, it increases 
the efficiency of phosphorylation, and it is critical for protein kinase A 
recognition in the context of the phospholamban pentamer. Given the synergistic 
consequences on contractility, it is not surprising that the mutants cause 
lethal, hereditary dilated cardiomyopathy.