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This Article Full Text Full Text (PDF) All Versions of this Article: 288/5/H2342    most recent 01133.2004v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (9) Citing Articles via Google Scholar Google Scholar Articles by Song, J. Articles by Cheung, J. Y. Search for Related Content PubMed PubMed Citation Articles by Song, J. Articles by Cheung, J. Y.

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

¹Department of Cellular and Molecular Physiology and ²Department of Medicine, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey; ³Weis Center for Research, Geisinger Medical Center, Danville, Pennsylvania; and ⁴Cardiovascular 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 Ca²⁺ concentration ([Ca²⁺]_i) homeostasis and inhibited Na⁺/Ca²⁺ 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, [Ca²⁺]_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 Ca²⁺-ATPase, Na⁺-K⁺-ATPase, and calsequestrin in adult rat myocytes. A COOH-terminal deletion mutant in which all four potential phosphorylation sites (Ser⁶², Ser⁶³, Ser⁶⁸, and Thr⁶⁹) were deleted, a partial COOH-terminal deletion mutant in which Ser⁶⁸ and Thr⁶⁹ 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 Ser⁶⁸ or Thr⁶⁹ in mediating PLM's effect on cardiac contractility. Focusing on Ser⁶⁸, the Ser⁶⁸ to Glu mutant was fully effective, the Ser⁶³ to Ala (leaving Ser⁶⁸ intact) mutant was partially effective, and the Ser⁶⁸ to Ala mutant was completely ineffective in modulating cardiac contractility, [Ca²⁺]_i transients, and NCX1 currents. Both the Ser⁶³ to Ala and Ser⁶⁸ to Ala mutants, as well as PLM, were able to coimmunoprecipitate NCX1. It is known that Ser⁶⁸ in PLM is phosphorylated by both protein kinases A and C. We conclude that regulation of cardiac contractility, [Ca²⁺]_i transients, and NCX1 activity by PLM is critically dependent on Ser⁶⁸. We suggest that PLM phosphorylation at Ser⁶⁸ may be involved in cAMP- and/or protein kinase C-dependent regulation of cardiac contractility.

adult rat myocyte culture; patch-clamp; fura-2; edge detection; excitation-contraction coupling

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