Serine 68 phosphorylation of phospholemman: acute isoform-specific activation of cardiac Na/K ATPase

Benjamin d. Z. Silverman, William Fuller, Philip Eaton, Juelin Deng, J. Randall Moorman, Joseph Y. Cheung, Andrew F. James, Michael J. Shattock

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    Abstract

    Objective: The mechanism by which the cardiac Na/K ATPase (NKA) is regulated by phosphorylation is controversial. We have used the perforated-patch technique to limit cell dialysis and maintain conditions as near physiological as possible. Methods: NKA pump current (Ip) was measured in isolated guinea pig ventricular myocytes, and its components (Ia1 and Ia2) defined by their differing dihydroouabain sensitivities. Results: Treatment with 1 µmol/l forskolin for 4 min at 35 °C caused a significant increase in Ia1 of 36 ± 15% (P<0.05, n=6), but no change in Ia2. The presence of the PKA selective inhibitor H89 (50 µmol/l) throughout the protocol blocked the effect of the forskolin on Ia1. Treatment with H89 alone did not change Ia1 or Ia2. Isoelectric focusing gels of the NKA a1 subunit demonstrated six charge states, which were unaltered following treatment with forskolin. Western blots using an antibody specific for the PKA phosphorylation consensus site on the a1 subunit showed no change in the phosphorylation status of this residue following forskolin treatment. The sarcolemmal protein phospholemman (PLM) was found associated with NKA a1 but not a2 subunits by immunoprecipitation and immunofluorescence. PLM was phosphorylated at serine 68, but not 63, following treatment with forskolin. Conclusions: PKA-dependent, a1-specific NKA activation may be mediated through phosphorylation of the accessory protein PLM, rather than direct a1 subunit phosphorylation.
    Original languageEnglish
    Pages (from-to)93-103
    Number of pages11
    JournalCardiovascular Research
    Volume65
    Issue number1
    DOIs
    Publication statusPublished - Jan 2005

    Keywords

    • Ion pumps
    • Na/K pump
    • Protein kinase A
    • Protein phosphorylation

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    Silverman, B. D. Z., Fuller, W., Eaton, P., Deng, J., Moorman, J. R., Cheung, J. Y., James, A. F., & Shattock, M. J. (2005). Serine 68 phosphorylation of phospholemman: acute isoform-specific activation of cardiac Na/K ATPase. Cardiovascular Research, 65(1), 93-103. https://doi.org/10.1016/j.cardiores.2004.09.005