Serine 68 of phospholemman is critical in modulation of contractility, [Ca2+]i transients, and Na+/Ca2+ exchange in adult rat cardiac myocytes

• Published in: American journal of physiology. Heart and circulatory physiology 2005-05, Vol.288 (5), p.H2342
• Main Authors: Song, Jianliang, Zhang, Xue-Qian, Ahlers, Belinda A, Carl, Lois L, Wang, JuFang, Rothblum, Lawrence I, Stahl, Richard C, Mounsey, J. Paul, Tucker, Amy L, Moorman, J. Randall, Cheung, Joseph Y
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Monoclonal - pharmacology; Calcium - metabolism; Cells, Cultured; Dogs; Humans; Kidney - cytology; Male; Membrane Proteins - genetics; Membrane Proteins - immunology; Membrane Proteins - metabolism; Mutagenesis; Myocardial Contraction - physiology; Myocytes, Cardiac - cytology; Myocytes, Cardiac - metabolism; Patch-Clamp Techniques; Phosphoproteins - genetics; Phosphoproteins - immunology; Phosphoproteins - metabolism; Rats; Rats, Sprague-Dawley; Sarcolemma - metabolism; Serine - metabolism; Sodium - metabolism; Sodium-Calcium Exchanger - metabolism
• ISSN: 0363-6135; 1522-1539
• DOI: 10.1152/ajpheart.01133.2004

1 Department of Cellular and Molecular Physiology and 2 Department of Medicine, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey; 3 Weis Center for Research, Geisinger Medical Center, Danville, Pennsylvania; and 4 Cardiovascular Division, Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville, Virginia Submitted 10 November 2004 ; accepted in final form 6 January 2005 Overexpression of phospholemman (PLM) in normal adult rat cardiac myocytes altered contractile function and cytosolic Ca 2+ concentration ([Ca 2+ ] i ) homeostasis and inhibited Na + /Ca 2+ exchanger (NCX1). In addition, PLM coimmunoprecipitated and colocalized with NCX1 in cardiac myocyte lysates. In this study, we evaluated whether the cytoplasmic domain of PLM is crucial in mediating its effects on contractility, [Ca 2+ ] i transients, and NCX1 activity. Canine PLM or its derived mutants were overexpressed in adult rat myocytes by adenovirus-mediated gene transfer. Confocal immunofluorescence images using canine-specific PLM antibodies demonstrated that the exogenous PLM or its mutants were correctly targeted to sarcolemma, t-tubules, and intercalated discs, with little to none detected in intracellular compartments. Overexpression of canine PLM or its mutants did not affect expression of NCX1, sarco(endo)plasmic reticulum Ca 2+ -ATPase, Na + -K + -ATPase, and calsequestrin in adult rat myocytes. A COOH-terminal deletion mutant in which all four potential phosphorylation sites (Ser 62 , Ser 63 , Ser 68 , and Thr 69 ) were deleted, a partial COOH-terminal deletion mutant in which Ser 68 and Thr 69 were deleted, and a mutant in which all four potential phosphorylation sites were changed to alanine all lost wild-type PLM's ability to modulate cardiac myocyte contractility. These observations suggest the importance of Ser 68 or Thr 69 in mediating PLM's effect on cardiac contractility. Focusing on Ser 68 , the Ser 68 to Glu mutant was fully effective, the Ser 63 to Ala (leaving Ser 68 intact) mutant was partially effective, and the Ser 68 to Ala mutant was completely ineffective in modulating cardiac contractility, [Ca 2+ ] i transients, and NCX1 currents. Both the Ser 63 to Ala and Ser 68 to Ala mutants, as well as PLM, were able to coimmunoprecipitate NCX1. It is known that Ser 68 in PLM is phosphorylated by both protein kinases A and C. We conclude that regulation of cardiac contractility, [Ca 2+ ] i transients, and NCX1 activ