Functional Interactions of Phospholemman (PLM) (FXYD1) with Na+,K+-ATPase

Human FXYD1 (phospholemman, PLM) has been expressed in Pichia pastoris with porcine α1/His10-β1 subunits of Na+,K+-ATPase or alone. Dodecyl-β-maltoside-soluble complexes of α1/β1/PLM have been purified by metal chelate chromatography, either from membranes co-expressing α1,His10-β1, and PLM or by in vitro reconstitution of PLM with α1/His10-β1 subunits. Comparison of functional properties of purified α1/His10-β1 and α1/His10-β1/PLM complexes show that PLM lowered K0.5 for Na+ ions moderately (≈30%) but did not affect the turnover rate or Km of ATP for activating Na+,K+-ATPase activity. PLM also stabilized the α1/His10-β1 complex. In addition, PLM markedly (>3-fold) reduced the K0.5 of Na+ ions for activating Na+-ATPase activity. In membranes co-expressing α1/His10-β1 with PLM the K0.5 of Na+ ions was also reduced, compared with the control, excluding the possibility that detergent or lipid in purified complexes compromise functional interactions. When expressed in HeLa cells with rat α1, rat PLM significantly raised the K0.5 of Na+ ions, whereas for a chimeric molecule consisting of transmembranes segments of PLM and extramembrane segments of FXYD4, the K0.5 of Na+ ions was significantly reduced, compared with the control. The opposite functional effects in P. pastoris and HeLa cells are correlated with endogenous phosphorylation of PLM at Ser68 or unphosphorylated PLM, respectively, as detected with antibodies, which recognize PLM phosphorylated at Ser68 (protein kinase A site) or unphosphorylated PLM. We hypothesize that PLM interacts with α1/His10-β1 subunits at multiple locations, the different functional effects depending on the degree of phosphorylation at Ser68. We discuss the role of PLM in regulation of Na+,K+-ATPase in cardiac or skeletal muscle cells.