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

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. NKA pump current (I(p)) was measured in isolated guinea pig ventricular myocytes, and its components (I(alpha 1) and I(alpha 2)) defined by their differing dihydroouabain sensitivities. Treatment with 1 micromol/l forskolin for 4 min at 35 degrees C caused a significant increase in I(alpha1) of 36+/-15% (P<0.05, n=6), but no change in I(alpha2). The presence of the PKA selective inhibitor H89 (50 micromol/l) throughout the protocol blocked the effect of the forskolin on I(alpha1). Treatment with H89 alone did not change I(alpha 1) or I(alpha 2). Isoelectric focusing gels of the NKA alpha1 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 alpha1 subunit showed no change in the phosphorylation status of this residue following forskolin treatment. The sarcolemmal protein phospholemman (PLM) was found associated with NKA alpha 1 but not alpha 2 subunits by immunoprecipitation and immunofluorescence. PLM was phosphorylated at serine 68, but not 63, following treatment with forskolin. PKA-dependent, alpha 1-specific NKA activation may be mediated through phosphorylation of the accessory protein PLM, rather than direct alpha1 subunit phosphorylation.