FXYD proteins are a family of seven small regulatory proteins, expressed in a tissue-specific manner, that associate with Na,KATPase 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 (␥ 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 (␣1␤1) to form ␣1␤1FXYD complexes. Compared with the control (␣1␤1), 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 7 E 2 conformational changes and cation occlusion, and FXYD1 protects strongly. Incubation of ␣1␤1 or ␣1␤1FXYD 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 ␣ subunit and the FXYD protein, which are in proximity. FXYD1, FXYD2, and FXYD4 co-expressed in HeLa cells with rat ␣1 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.

The human α(1)/His(10)-β(1) isoform of the Na,K-ATPase has been expressed in Pichia pastoris, solubilized in n-dodecyl-β-maltoside, and purified by metal chelate chromatography. The α(1)β(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)-β(1) complex and stabilizes it in an active mode. The functional properties of the α(1)/His(10)-β(1) and α(1)/His(10)-β(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)β(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.

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) 7 E 2 P and E 2 (2K) 3 E 1 Na transitions in native Na ؉ ,K ؉ -ATPase.The kinetic mechanism of P-type cation pumps is now well established. Active cation transport involves covalent phosphorylation by ATP and dephosphorylation of an aspartate residue coupled to cation movements mediated by E 1 7 E 2 2 conformational changes. The molecular mechanism is, of course, the central question of energy transduction. Molecular structures of the sarcoplasmic reticulum Ca 2ϩ -ATPase (SERCA1) in several conformations are available (1-3). Two crystal structures of the Na ؉ ,K ؉ -ATPase, consisting of ␣, ␤, and FYXD subunits (4), in an E 2 (2Rb)-MgF 4 2Ϫ conformation (equivalent to E 2 (2Rb)⅐P) have been published recently (5, 6). The architecture of the ␣ subunit is very similar to the Ca 2ϩ -ATPase, consisting of head, stalk, and membrane sectors, with 10 trans-membrane segments and a cytoplasmic sector consisting of N (nucleotide binding), P (phosphorylating), and A (anchor or actuator) domains. Compared with the first published structure of the Na ϩ ,K ϩ -ATPase at 3.5 Å (5), the more recent structure at 2.4 Å (6) reveals greater detail of the cation binding domain, resolution of the ␤ subunit ectodomain, and an FXYD protein ectodomain.In the case of Na ϩ ,K ϩ -ATPase, three Na ؉ ions and two K ؉ ions are transported in the E 1 ATP 3 E 1 P(3Na) 3E 2 P and the E 2 P 3 E 2 (2K) 3 E 1 ATP halves of the cycle, respectively, at the expense of one molecule of ATP. Binding of ATP with low affinity to E 2 (2K) is the first step in the catalytic cycle and is followed by accelerated conversion of E 2 (2K)ATP to E 1 ⅐ATP in which ATP is bound with high affinity (7-9). In the case of Ca 2ϩ -ATPase, two Ca 2ϩ ions and two protons are transported in the E 1 ATP 3 E 1 P 3 E 2 P E 2 P 3 E 2 3 E 1 ATP halves of the cycle, respectively.The mechanism of energy transduction by P-type cation pumps is best addressed by reference to the Ca 2ϩ -ATPase, which has been crystallized in most of the relevant conformations, reviewed recently in detail (1-3). Crystal structures of Ca 2ϩ -ATPase show mainly rigid body movements of the N, P, and A domains, mediated by the flexible linkers between the N and P and between the A domain and trans-membrane segment (M1-M3), coupled mechanically to movements of M1-M6 trans-membrane segments that allow alternate access of two Ca 2ϩ ions and two protons to the occlusion sites within M4, M5, M6, and M8. M7-M10 segments appear to act as immovable anchors. In the E 1 ATP state, N and P domains are in close proximity cross-linked by the bound ATP, whereas the A domain is displaced to one side. Following phosphorylation to E 1 P, the conformational change to E 2 P involves a large rotation of the A domain bringing it into close proximity with th...