Identification of an Endogenous Inhibitor of the Cardiac Na+/Ca2+ Exchanger, Phospholemman

• Published in: The Journal of biological chemistry 2005-05, Vol.280 (20), p.19875-19882
• Main Authors: Ahlers, Belinda A., Zhang, Xue-Qian, Moorman, J. Randall, Rothblum, Lawrence I., Carl, Lois L., Song, Jianliang, Wang, JuFang, Geddis, Lisa M., Tucker, Amy L., Mounsey, J. Paul, Cheung, Joseph Y.
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Animals; Base Sequence; Cell Line; Cell Membrane - metabolism; DNA - genetics; Humans; In Vitro Techniques; Male; Membrane Potentials; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - metabolism; Mutagenesis, Site-Directed; Myocardium - metabolism; Patch-Clamp Techniques; Phosphoproteins - chemistry; Phosphoproteins - genetics; Phosphoproteins - metabolism; Rats; Rats, Sprague-Dawley; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sodium-Calcium Exchanger - antagonists & inhibitors; Sodium-Calcium Exchanger - genetics; Sodium-Calcium Exchanger - metabolism; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M414703200

Rapid and precise control of Na+/Ca2+ exchanger (NCX1) activity is essential in the maintenance of beat-to-beat Ca2+ homeostasis in cardiac myocytes. Here, we show that phospholemman (PLM), a 15-kDa integral sarcolemmal phosphoprotein, is a novel endogenous protein inhibitor of cardiac NCX1. Using a heterologous expression system that is devoid of both endogenous PLM and NCX1, we first demonstrated by confocal immunofluorescence studies that both exogenous PLM and NCX1 co-localized at the plasma membrane. Reciprocal co-immunoprecipitation studies revealed specific protein-protein interaction between PLM and NCX1. The functional consequences of direct association of PLM with NCX1 was the inhibition of NCX1 activity, as demonstrated by whole-cell patch clamp studies to measure NCX1 current density and radiotracer flux assays to assess Na+-dependent 45Ca2+ uptake. Inhibition of NCX1 by PLM was specific, because a single mutation of serine 68 to alanine in PLM resulted in a complete loss of inhibition of NCX1 current, although association of the PLM mutant with NCX1 was unaltered. In native adult cardiac myocytes, PLM co-immunoprecipitated with NCX1. We conclude that PLM, a member of the FXYD family of small ion transport regulators known to modulate Na+-K+-ATPase, also regulates Na+/Ca2+ exchange in the heart.