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

Abstract

The human a 1 /His 10 -b 1 isoform of Na,K-ATPase 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 (K 1/2 ) 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 negatively charged lipid DOPS (\10 %), FXYD1 increases K 1/2 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 concentrations of DOPS ([10 %), little or no effect of FXYD1 on the K 1/2 of Na ? ions is observed. Depending on ionic strength and lipid composition of the proteoliposomes, FXYD1 can alter the K 1/2 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 interact with the cytoplasmic N domain of the a-subunit, the interaction being strongly affected by phosphorylation at Ser68 and the surface charge of the membrane.

Molecular Mechanisms and Kinetic Effects of FXYD1 and Phosphomimetic Mutants on Purified Human Na,K-ATPase

“…Due to the lower solubility of such lipid mixtures in DDM only, a solution of 1% DDM and 1% C 12 E 8 in Buffer H (25 mM imidazole, 1 mM EDTA, 5 mM MgSO 4 , 5 mM Na 2 SO 4 , 70 mM K 2 SO 4 , pH 7.2) was chosen to solubilize the lipids. Proteoliposome formation containing recombinant ␣1His 10 -␤1FXYD1 complexes was performed as described previously (24). In short, to obtain a lipid concentration of 8 mg/ml, the desired lipid mixture was dissolved in Buffer H with 2% DDM.…”

“…Experiments were performed corresponding to a recent study from this laboratory (24). In short, 1 ml of buffer solutions containing 25 mM imidazole, 1 mM EDTA, 2.5 mM MgSO 4 , and various concentrations of Na 2 SO 4 , pH 7.2, were thermally equilibrated in a cuvette equipped with a magnetic stirrer.…”

“…The initial slope of the fluorescence increase after ATP addition, dF norm (t)/dt, corresponding to the increase in membrane potential, is proportional to the product of the pumping rate and the amount of active pump molecules, averaged over all vesicles in the solution (26). The initial slope was determined as described recently (24) and used as parameter to determine the pump rate. Its dependence on the extravesicular Na ϩ concentration has to be determined for each series with the same vesicle preparation and can be exploited to evaluate the K 0.…”

“…We have shown recently that after reconstitution of the detergent-soluble recombinant Na,K-ATPase into proteoliposomes, generation of the pump-mediated electrogenic potential is conveniently detected using the voltage-sensitive dye, oxonol VI (24). These experiments allow determination of the half-saturating Na ϩ concentration, K 0.5 Na for generation of the electrogenic potential accompanying Na,K-ATPase activity with and without FXYD and the three phosphomimetic mutants.…”

“…The interaction of the transmembrane helices has been proven in detergent-solubilized isolated complexes and in native cell membrane and is important for maintaining stability of the protein (22,23). In recent studies, it has been shown that the charge of the cytoplasmic domain plays an important role in the regulation of the Na,K-ATPase (24). The strongly positively charged H4 helix of FXYD1 is assumed to adhere to a negatively charged region in the cytoplasmic domain of the Na,K-ATPase when unphosphorylated.…”