Regulation of cardiac Na+/Ca2+ exchanger by phospholemman

Phospholemman (PLM) is the first sequenced member of the FXYD family of regulators of ion transport. The mature protein has 72 amino acids and consists of an extracellular N terminus containing the signature FXYD motif, a single transmembrane (TM) domain, and a cytoplasmic C-terminal domain containing four potential sites for phosphorylation. PLM and other members of the FXYD family are known to regulate Na+-K+-ATPase. Using adenovirus-mediated gene transfer into adult rat cardiac myocytes, we showed that changes in contractility and intracellular Ca2+ homeostasis associated with PLM overexpression or downregulation are not consistent with the effects expected from inhibition of Na+-K+-ATPase by PLM. Additional studies with heterologous expression of PLM and cardiac Na+/Ca2+ exchanger 1 (NCX1) in HEK293 cells and cardiac myocytes isolated from PLM-deficient mice demonstrated by co-localization, co-immunoprecipitation, and electrophysiological and radioactive tracer uptake techniques that PLM associates with NCX1 in the sarcolemma and transverse tubules and that PLM inhibits NCX1, independent of its effects on Na+-K+-ATPase. Mutational analysis indicates that the cytoplasmic domain of PLM is required for its regulation of NCX1. In addition, experiments using phosphomimetic and phospho-deficient PLM mutants, as well as activators of protein kinases A and C, indicate that PLM phosphorylated at serine68 is the active form that inhibits NCX1. This is in sharp contrast to the finding that the unphosphorylated PLM form inhibits Na+-K+-ATPase. We conclude that PLM regulates cardiac contractility by modulating the activities of NCX and Na+-K+-ATPase.