Phospholemman (FXYD1) raises the affinity of the human α1ß1 isoform of Na,K-ATPase for Na ions.

The human α(1)/His(10)-ß(1) isoform of the Na,K-ATPase has been expressed in Pichia pastoris, solubilized in n-dodecyl-ß-maltoside, and purified by metal chelate chromatography. The α(1)ß(1) complex spontaneously associates in vitro with the detergent-solubilized purified human FXYD1 (phospholemman) expressed in Escherichia coli. It has been confirmed that FXYD1 spontaneously associates in vitro with the α(1)/His(10)-ß(1) complex and stabilizes it in an active mode. The functional properties of the α(1)/His(10)-ß(1) and α(1)/His(10)-ß(1)/FXYD1 complexes have been investigated by fluorescence methods. The electrochromic dye RH421 which monitors binding to and release of ions from the binding sites has been applied in equilibrium titration experiments to determine ion binding affinities and revealed that FXYD1 induces an ∼30% increase of the Na(+)-binding affinity in both the E(1) and P-E(2) conformations. By contrast, it does not affect the affinities for K(+) and Rb(+) ions. Phosphorylation induced partial reactions of the enzyme have been studied as backdoor phosphorylation by inorganic phosphate and in kinetic experiments with caged ATP in order to evaluate the ATP-binding affinity and the time constant of the conformational transition, Na(3)E(1)-P → P-E(2)Na(3). No significant differences with or without FXYD1 could be detected. Rate constants of the conformational transitions Rb(2)E(1) → E(2)(Rb(2)) and E(2)(Rb(2)) → Na(3)E(1), investigated with fluorescein-labeled Na,K-ATPase, showed only minor or no effects of FXYD1, respectively. The conclusion from all these experiments is that FXYD1 raises the binding affinity of α(1)ß(1) for Na ions, presumably at the third Na-selective binding site. In whole cell expression studies FXYD1 reduces the apparent affinity for Na ions. Possible reasons for the difference from this study using the purified recombinant Na,K-ATPase are discussed.

FXYD proteins stabilize Na,K-ATPase: amplification of specific phosphatidylserine-protein interactions.

FXYD proteins are a family of seven small regulatory proteins, expressed in a tissue-specific manner, that associate with Na,K-ATPase as subsidiary subunits and modulate kinetic properties. This study describes an additional property of FXYD proteins as stabilizers of Na,K-ATPase. FXYD1 (phospholemman), FXYD2 (γ subunit), and FXYD4 (CHIF) have been expressed in Escherichia coli and purified. These FXYD proteins associate spontaneously in vitro with detergent-soluble purified recombinant human Na,K-ATPase (α1ß1) to form α1ß1FXYD complexes. Compared with the control (α1ß1), all three FXYD proteins strongly protect Na,K-ATPase activity against inactivation by heating or excess detergent (C(12)E(8)), with effectiveness FXYD1 > FXYD2 ≥ FXYD4. Heating also inactivates E(1) ↔ E(2) conformational changes and cation occlusion, and FXYD1 protects strongly. Incubation of α1ß1 or α1ß1FXYD complexes with guanidinium chloride (up to 6 m) causes protein unfolding, detected by changes in protein fluorescence, but FXYD proteins do not protect. Thus, general protein denaturation is not the cause of thermally mediated or detergent-mediated inactivation. By contrast, the experiments show that displacement of specifically bound phosphatidylserine is the primary cause of thermally mediated or detergent-mediated inactivation, and FXYD proteins stabilize phosphatidylserine-Na,K-ATPase interactions. Phosphatidylserine probably binds near trans-membrane segments M9 of the α subunit and the FXYD protein, which are in proximity. FXYD1, FXYD2, and FXYD4 co-expressed in HeLa cells with rat α1 protect strongly against thermal inactivation. Stabilization of Na,K-ATPase by three FXYD proteins in a mammalian cell membrane, as well the purified recombinant Na,K-ATPase, suggests that stabilization is a general property of FXYD proteins, consistent with a significant biological function.

Neutralization of the charge on Asp 369 of Na+,K+-ATPase triggers E1 <--> E2 conformational changes.

This work investigates the role of charge of the phosphorylated aspartate, Asp(369), of Na(+),K(+)-ATPase on E(1) <--> E(2) conformational changes. Wild type (porcine alpha(1)/His(10)-beta(1)), D369N/D369A/D369E, and T212A mutants were expressed in Pichia pastoris, labeled with fluorescein 5'-isothiocyanate (FITC), and purified. Conformational changes of wild type and mutant proteins were analyzed using fluorescein fluorescence (Karlish, S. J. (1980) J. Bioenerg. Biomembr. 12, 111-136). One central finding is that the D369N/D369A mutants are strongly stabilized in E(2) compared with wild type and D369E or T212A mutants. Stabilization of E(2)(Rb) is detected by a reduced K(0.5)Rb for the Rb(+)-induced E(1) <--> E(2)(2Rb) transition. The mechanism involves a greatly reduced rate of E(2)(2Rb) --> E(1)Na with no effect on E(1) --> E(2)(2Rb). Lowering the pH from 7.5 to 5.5 strongly stabilizes wild type in E(2) but affects the D369N mutant only weakly. Thus, this "Bohr" effect of pH on E(1) <--> E(2) is due largely to protonation of Asp(369). Two novel effects of phosphate and vanadate were observed with the D369N/D369A mutants as follows. (a) E(1) --> E(2).P is induced by phosphate without Mg(2+) ions by contrast with wild type, which requires Mg(2+). (b) Both phosphate and vanadate induce rapid E(1) --> E(2) transitions compared with slow rates for the wild type. With reference to crystal structures of Ca(2+)-ATPase and Na(+),K(+)-ATPase, negatively charged Asp(369) favors disengagement of the A domain from N and P domains (E(1)), whereas the neutral D369N/D369A mutants favor association of the A domain (TGES sequence) with P and N domains (E(2)). Changes in charge interactions of Asp(369) may play an important role in triggering E(1)P(3Na) <--> E(2)P and E(2)(2K) --> E(1)Na transitions in native Na(+),K(+)-ATPase.