Isoform Specificity of the Na/K-ATPase Association and Regulation by Phospholemman

Phospholemman (PLM) phosphorylation mediates enhanced Na/K-ATPase (NKA) function during adrenergic stimulation of the heart. Multiple NKA isoforms exist, and their function/regulation may differ. We combined fluorescence resonance energy transfer (FRET) and functional measurements to investigate isoform specificity of the NKA-PLM interaction. FRET was measured as the increase in the donor fluorescence (CFP-NKA-alpha 1 or CFP-NKA-alpha 2) during progressive acceptor (PLM-YFP) photobleach in HEK-293 cells. Both pairs exhibited robust FRET (maximum of 23.6 +/- 3.4% for NKA-alpha 1 and 27.5 +/- 2.5% for NKA-alpha 2). Donor fluorescence depended linearly on acceptor fluorescence, indicating a 1: 1 PLM: NKA stoichiometry for both isoforms. PLM phosphorylation induced by cAMP-dependent protein kinase and protein kinase C activation drastically reduced the FRET with both NKA isoforms. However, submaximal cAMP-dependent protein kinase activation had less effect on PLM-NKA-alpha 2 versus PLM-NKA-alpha 1. Surprisingly, ouabain virtually abolished NKA-PLM FRET but only partially reduced co-immunoprecipitation. PLM-CFP also showed FRET to PLM-YFP, but the relationship during progressive photobleach was highly nonlinear, indicating oligomers involving >= 3 monomers. Using cardiac myocytes from wild-type mice and mice where NKA-alpha 1 is ouabain-sensitive and NKA-alpha 2 is ouabain-resistant, we assessed the effects of PLM phosphorylation on NKA-alpha 1 and NKA-alpha 2 function. Isoproterenol enhanced internal Na+ affinity of both isoforms (K-1/2 decreased from 18.1 +/- 2.0 to 11.5 +/- 1.9 mM for NKA-alpha 1 and from 16.4 +/- 2.5 to 10.4 +/- 1.5 mM for NKA-alpha 2) without altering maximum transport rate (V-max). Protein kinase C activation also decreased K-1/2 for both NKA-alpha 1 and NKA-alpha 2 (to 9.4 +/- 1.0 and 9.1 +/- 1.1 mM, respectively) but increased V-max only for NKA-alpha 2 (1.9 +/- 0.4 versus 1.2 +/- 0.5 mM/min). In conclusion, PLM associates with and modulates both NKA-alpha 1 and NKA-alpha 2 in a comparable but not identical manner.