Serine 68 phosphorylation of phospholemman: acute isoform-specific activation of cardiac Na/K ATPase.
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(alpha 1) and I(alpha 2)) defined by their differing dihydroouabain sensitivities.


RESULTS
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.


CONCLUSIONS
PKA-dependent, alpha 1-specific NKA activation may be mediated through phosphorylation of the accessory protein PLM, rather than direct alpha1 subunit phosphorylation.
Silverman
Benjamin d Z
Bd
Cardiovascular Division, King's College London, The Rayne Institute, St Thomas' Hospital, London SE1 7EH, UK.
Fuller
William
W
Eaton
Philip
P
Deng
Juelin
J
Moorman
J Randall
JR
Cheung
Joseph Y
JY
James
Andrew F
AF
Shattock
Michael J
MJ
D000818:Animals; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D004789:Enzyme Activation; D006168:Guinea Pigs; D006352:Heart Ventricles; D007546:Isoquinolines / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D032383:Myocytes, Cardiac / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D020033:Protein Isoforms / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013449:Sulfonamides / Q000494:pharmacology
D007546:Isoquinolines; D008565:Membrane Proteins; D010750:Phosphoproteins; D020033:Protein Isoforms; D013449:Sulfonamides; C069141:phospholemman; D005576:Colforsin; D012694:Serine; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D000254:Sodium-Potassium-Exchanging ATPase; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide