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. MultipleNKAisoforms exist, and their function/regulation may differ. We combined fluorescence resonance energy transfer (FRET) and functional measurements to investigate isoform specificity of theNKA-PLMinteraction. FRET was measured as the increase in the donor fluorescence (CFP-NKA-α1 or CFPNKA-α2) during progressive acceptor (PLM-YFP) photobleach in HEK-293 cells. Both pairs exhibited robust FRET (maximum of 23.6 ± 3.4% for NKA-α1 and 27.5 ± 2.5% for NKA-α2). 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-α2 versus PLMNKA-α1. Surprisingly, ouabain virtually abolished NKA-PLM FRET but only partially reduced co-immunoprecipitation. PLMCFP 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-α1 is ouabain-sensitive and NKA-α2 is ouabain-resistant, we assessed the effects ofPLMphosphorylation on NKA-α1 and NKA-α2 function. Isoproterenol enhanced internal Na⁺ affinity of both isoforms (K_1/2 decreased from 18.1±2.0 to 11.5±1.9mM for NKA-α1 and from 16.4±2.5 to 10.4 ± 1.5 mM for NKA-α2) without altering maximum transport rate (V_max). Protein kinase C activation also decreased K_1/2 for both NKA-α1 and NKA-α2 (to 9.4 ± 1.0 and 9.1 ± 1.1 mM, respectively) but increasedVmax only forNKA-α2 (1.9±0.4 versus 1.2±0.5 mM/min). In conclusion, PLM associates with and modulates both NKA-α1 andNKA-α2 in a comparable but not identical manner.