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

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 (I_p) was measured in isolated guinea pig ventricular myocytes, and its components (I_α1 and I_α2) defined by their differing dihydroouabain sensitivities. Results: Treatment with 1 μmol/l forskolin for 4 min at 35 °C caused a significant increase in I_α1 of 36 ± 15% (Pn=6), but no change in I_α2. The presence of the PKA selective inhibitor H89 (50 μmol/l) throughout the protocol blocked the effect of the forskolin on I_α1. Treatment with H89 alone did not change I_α1 or I_α2. Isoelectric focusing gels of the NKA α1 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 α1 subunit showed no change in the phosphorylation status of this residue following forskolin treatment. The sarcolemmal protein phospholemman (PLM) was found associated with NKA α1 but not α2 subunits by immunoprecipitation and immunofluorescence. PLM was phosphorylated at serine 68, but not 63, following treatment with forskolin. Conclusions: PKA-dependent, α1-specific NKA activation may be mediated through phosphorylation of the accessory protein PLM, rather than direct α1 subunit phosphorylation.