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1 Cellular & Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, PA, USA; Weis Center for Research, Geisinger Medical Center, Danville, PA, USA
2 Weis Center for Research, Geisinger Medical Center, Danville, PA, USA
3 Internal Medicine, University of Virginia, Charlottesville, VA, USA
4 Cellular & Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, PA, USA; Medicine, Pennsylvania State Univeristy College of Medicine, Hershey, PA, USA; Weis Center for Research, Geisinger Medical Center, Danville, PA, USA
* To whom correspondence should be addressed. E-mail: jyc1{at}psu.edu.
Overexpression of phospholemman (PLM) in normal adult rat cardiac myocytes has been shown to alter contractile function and cytosolic Ca2+ concentration ([Ca2+]i) homeostasis, and inhibit Na+/Ca2+ exchanger (NCX1) function. In addition, PLM co-immunoprecipitated with NCX1 in cardiac myocyte lysates. In this study, we evaluated which part of the PLM molecule is crucial in mediating its effects on contractility, [Ca2+]i transients and NCX1 activity. Canine PLM or its derived mutants was overexpressed in adult rat myocytes by adenovirus mediated gene transfer. Confocal immunofluorescence images using canine-specific PLM antibodies demonstrated that the exogenous PLM or canine PLM mutants was 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, Na+-K+-ATPase, or sarco(endo)plasmic reticulum Ca2+-ATPase in adult rat myocytes. A C-terminal deletion mutant in which all 4 potential phosphorylation sites (Ser62, Ser63, Ser68, Thr69) were deleted, a partial C-terminal deletion mutant in which Ser68 and Thr69 were deleted, and a mutant in which all four potential phosphorylation sites (Ser62, Ser63, Ser68, Thr69) were changed to alanine, all lost the ability to modulate cardiac myocyte contractility. These observations suggest the importance of Ser68 or Thr69 in mediating the effect of PLM on cardiac contractility. Focusing on Ser68, Ser68-to-Glu mutant was fully effective, Ser63-to-Ala (leaving Ser68 intact) mutant was partially effective, and Ser68-to-Ala mutant was completely ineffective in modulating cardiac contractility, [Ca2+]i transients, and NCX1 currents. Both Ser63-to-Ala and Ser68-to-Ala mutants, as well as PLM, were able to co-immunoprecipitate NCX1. It is known that Ser68 in PLM is phosphorylated by both protein kinase 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.
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