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Expression of phospholamban in C2C12 cells and regulation of endogenous SERCA1 activity

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Abstract

Phospholamban (PLB) is a regulator of the sarcoplasmic reticulum Ca2+-ATPase (SERCA2) expressed in cardiac, slow-twitch skeletal, and smooth muscles. Phospholamban is not expressed in the sarcoplasmic reticulum of fast-twitch skeletal muscle, but it can regulate the sarcoplasmic reticulum Ca2+-ATPase activity (SERCA1) expressed in this muscle,in vitro. To determine whether phospholamban can regulate SERCA1 activity in its native membrane environment, phospholamban was stably transfected into a cell line (C2C12) derived from murine fast-twitch skeletal muscle. Differentiation of C2C12 myoblasts to myotubes was associated with induction of SERCA1 expression, assessed by Western blotting analysis using Ca2+-ATPase isoform specific antibodies. The expressed phospholamban protein was localized in the microsomal fraction isolated from C2C12 myotubes. To determine the effect of phospholamban expression on SERCA1 activity, microsomes were isolated from transfected and nontransfected C2C12 cell myotubes, and the initial rates of45Ca2+-uptake were determined over a wide range of Ca2+ concentrations (0.1–10 μM). Expression of phospholamban was associated with inhibition of the initial rates of Ca2+-uptake at low [Ca2+] and this resulted in a decrease in the affinity of SERCA1 for Ca2+ (0.27±0.02 μM in nontransfected vs. 0.41±0.03 μM in PLB transfected C2C12 cells). These findings indicate that phospholamban expression in C2C12 cells is associated within inhibition of the endogenous SERCA1 activity and provide evidence that phospholamban is capable of regulating this Ca2+-ATPase isoform in its native membrane environment.

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  1. Donald G. Ferguson

    Present address: Department of Anatomy, Case Western Reserve University, 10900 Euclid Avenue, 44106-4930, Cleveland, OH, USA

Authors and Affiliations

  1. Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, 231 Bethesda Avenue, 45267-057S, Cincinnati, Ohio, USA

    Judy M. Harrer, Sathivel Ponniah & Evangelia G. Kranias

  2. Department of Physiology and Biophys, University of Cincinnati College of Medicine, 231 Bethesda Avenue, 45267-057S, Cincinnati, Ohio, USA

    Donald G. Ferguson

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  1. Judy M. Harrer
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Harrer, J.M., Ponniah, S., Ferguson, D.G. et al. Expression of phospholamban in C2C12 cells and regulation of endogenous SERCA1 activity. Mol Cell Biochem 146, 13–21 (1995). https://doi.org/10.1007/BF00926876

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  • DOI: https://doi.org/10.1007/BF00926876

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