Cardiovascular Research

Cardiovascular Research 2005 65(1):93-103; doi:10.1016/j.cardiores.2004.09.005

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Copyright © 2004, European Society of Cardiology

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

Benjamin d.Z. Silverman^a,1, William Fuller^a,1, Philip Eaton^a, Juelin Deng^b, J. Randall Moorman^c, Joseph Y. Cheung^d, Andrew F. James^e and Michael J. Shattock^a,*

^aCardiovascular Division, King's College London, The Rayne Institute, St Thomas' Hospital, London SE1 7EH, UK
^bWest China Hospital, Sichuan University, People's Republic of China
^cDepartment of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA
^dWeis Center for Research, Geisinger Medical Center, Danville, PA 17822, USA
^eDepartment of Physiology, School of Medical Sciences, University of Bristol, Bristol, United Kingdom

^* Corresponding author. Tel.: +44 20 7188 0945; fax: +44 20 7188 3902. Email address: michael.shattock{at}kcl.ac.uk

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 (I_p) was measured in isolated guinea pig ventricular myocytes, and its components (I₁ and I₂) defined by their differing dihydroouabain sensitivities.

Results: Treatment with 1 µmol/l forskolin for 4 min at 35 °C caused a significant increase in I₁ of 36 ± 15% (P<0.05, n=6), but no change in I₂. The presence of the PKA selective inhibitor H89 (50 µmol/l) throughout the protocol blocked the effect of the forskolin on I₁. Treatment with H89 alone did not change I₁ or I₂. Isoelectric focusing gels of the NKA 1 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 1 subunit showed no change in the phosphorylation status of this residue following forskolin treatment. The sarcolemmal protein phospholemman (PLM) was found associated with NKA 1 but not 2 subunits by immunoprecipitation and immunofluorescence. PLM was phosphorylated at serine 68, but not 63, following treatment with forskolin.

Conclusions: PKA-dependent, 1-specific NKA activation may be mediated through phosphorylation of the accessory protein PLM, rather than direct 1 subunit phosphorylation.

KEYWORDS Ion pumps; Na/K pump; Protein kinase A; Protein phosphorylation

Abbreviations: NKA, Na/K ATPase • PLM, phospholemman • DIDS, 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid

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¹ These authors contributed equally to this work.

Time for primary review 18 days

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