Surface Charges of the Membrane Crucially Affect Regulation of Na,K-ATPase by Phospholemman (FXYD1)

Abstract

Abstract The human a1/His10-b1 isoform of Na,K-ATP-ase has been reconstituted as a complex with and without FXYD1 into proteoliposomes of various lipid compositions in order to study the effect of the regulatory subunit on the half-saturating Na? concentration (K1/2) of Na? ions for activation of the ion pump. It has been shown that the fraction of negatively charged lipid in the bilayer crucially affects the regulatory properties. At low concentrations of the nega-tively charged lipid DOPS (\10 %), FXYD1 increases K1/2 of Na? ions for activation of the ion pump. Phosphorylation of FXYD1 by protein kinase A at Ser68 abrogates this effect. Conversely, for proteoliposomes made with high concen-trations of DOPS ([10 %), little or no effect of FXYD1 on theK1/2 ofNa? ions is observed. Depending on ionic strength and lipid composition of the proteoliposomes, FXYD1 can alter the K1/2 of Na? ions by up to twofold. We propose possible molecular mechanisms to explain the regulatory effects of FXYD1 and the influence of charged lipid and protein phosphorylation. In particular, the positively charged C-terminal helix of FXYD1 appears to be highly mobile and may interactwith the cytoplasmicNdomain of thea-subunit, the interaction being strongly affected by phosphorylation at Ser68 and the surface charge of the membrane