FXYD1 Increases the Na + -binding Affinity in the E 1 and P-E 2 Conformations of the Na,K-ATPase

The kinetic properties of α1/His10-β1 and α1/His10-β1/FXYD1 determined with the meth-ods exploiting the dye RH421 are summarized in Table 10. Since comparable investigations have been performed only with membrane-bound Na,K-ATPase from rabbit kidney, the pa-rameters of the recombinant, purified, detergent-solubilized human preparations are com-pared with the respective values of the native Na,K-ATPase in membrane fragments. The equilibrium dissociation constants of the recombinant proteins are very similar to those of the native enzyme, especially in the case of the α₁/His₁₀-β₁/FXYD1 complex. The rabbit en-zyme consists of the heterotrimer α1/β1/FXYD2. FXYD2 is a regulatory subunit closely re-lated to FXYD1, but reported to decrease the Na⁺-binding affinity (52-58).

Native

Table 10. Comparison of the kinetic properties of the native membrane-bound Na,K-ATPase from rabbit kidney and of the recombinant, purified, detergent-solubilized human α1/His10-β1 and α1/His10 -β1/FXYD1 investigated with the dye RH421. The references for the data of the rabbit enzyme are given in parenthesis.

As discussed in 4.1, the detergent-solubilized α1/His10-β1 enzyme has a lower specific activity compared to the membrane-bound native Na,K-ATPase. The reason has been as-signed both to the modified lipid environment and to a lower number of active enzyme mol-ecules due to partial detergent-induced inactivation during the purification procedure. The

complex with FXYD1 also shows an ~ 50% lower specific ATPase activity compared to that of the membrane-bound native Na,K-ATPase, (3.2). Since FXYD1 has a strong stabilizing effect against detergent-mediated inactivation, as discussed in 4.2, such a difference is likely to arise mainly from a lower turnover rate of the recombinant enzymes in the solubilized en-vironment. Indeed, the time constant of the conformational transition (Na3)E1-P → P-E2Na3

is ~ 40% larger for the recombinant preparations compared to the membrane-bound native enzyme under similar conditions. In principle, this difference could also be due to the pres-ence of FXYD2 in the native enzyme; since the conformational transitions of the detergent-solubilized rabbit enzyme have not been investigated, it is not possible to assign this effect more precisely.

The comparison of the reaction steps of the Post-Albers cycle of the enzyme with and without FXYD1 reveals a single kinetic modification induced by FXYD1, an ~ 20-30% in-crease in Na⁺-binding affinity in both the E1 and P-E2 conformations. In contrast, the binding affinities for K⁺ and Rb⁺ ions are independent of the presence of FXYD1. All three ion spe-cies are able to bind to the two bifunctional ion-binding sites, but only sodium is able to oc-cupy the third ion-binding site. Therefore, the result that sodium binding, but not potassium and rubidium binding, is affected by the presence of FXYD1 indicates a specific effect of FXYD1 on the third, Na⁺-selective ion-binding site. The result fits with the proximity of FXYD proteins to helix M9 of the α subunit that is supposed to be involved in the binding of the third Na⁺ ion (29) (Fig. 64).

Figure 64. View from cytoplasmic surface of the transmembrane segments of the shark Na,K-ATPase, showing the interaction of FXYD10 with helix M9 of the α subunit (111).

As reported in 1.4.4, the current hypothesis about the mechanism of interaction of FXYD1 with the Na,K-ATPase is the following: the unphosphorylated FXYD1 may inhibit the enzyme by reducing the Na⁺- and, possibly, the K⁺-binding affinity, while FXYD1 phosphorylated by PKA may stimulate the ion pump by abolishing the effect on the Na⁺ -binding affinity. Since phosphorylation by PKA occurs at Ser 68, localized in the cytoplasmic segment of FXYD1, the effect on the Na⁺-binding affinity seems to be due to the interaction of the enzyme with this segment. However, experiments with a FXYD1/FXYD4 chimera containing the transmembrane segment of FXYD1 have shown

that this segment is responsible for an increase of about 20% in the intracellular Na⁺-binding affinity of the enzyme (74). Considering this, it is possible that the different segments of FXYD1 affect the Na⁺-binding affinity of the Na,K-ATPase in a distinct way: on the one hand, the unphosphorylated cytoplasmic segment could decrease the apparent Na⁺-binding affinity; on the other hand the transmembrane segment may increase the intrinsic Na⁺ -binding affinity. This second effect could be masked by the decrease induced by the unphosphorylated cytoplasmic segment and be revealed only upon phosphorylation of it, depending on the degree of phosphorylation. Therefore, the effect of FXYD1 on the Na⁺ -binding affinity detected in the current study could be explained by the interaction of the enzyme with only the transmembrane segment of FXYD1. The interaction with the cytoplasmic segment of FXYD1 may be somehow missing.

A higher apparent Na⁺-binding affinity of the detergent-solubilized recombinant α1/His10-β1/FXYD1 compared to α1/His10-β1 has been already detected in Na,K-ATPase ac-tivity assays (74). In this paper, it has been observed that the purified, detergent-solubilized recombinant α1/His10-β1/FXYD1 complex obtained by in vitro reconstitution after separate expression in P. pastoris has the same functional properties of the purified, detergent-solubilized recombinant complex obtained after co-expression in the yeast membrane. As a consequence, it can be excluded that the α1/β1/FXYD1 association during the in vitro recon-stitution is improper in some way. In (74), FXYD1 expressed in P. pastoris has been found to be partially phosphorylated at Ser 68. Thus, the higher Na⁺-binding affinity in the pres-ence of FXYD1 observed with the detergent-solubilized preparations in such study has been related to the phosphorylated state of FXYD1. However, in the current study FXYD1 has been expressed in E. coli, where it is not phosphorylated (112). Therefore, phosphorylation cannot be made responsible for the observed increase in Na⁺-binding affinity. The fact that the detergent-solubilized recombinant α1/His10-β1/FXYD1 preparations show an increase in Na⁺-binding affinity independently of the phosphorylation state of FXYD1 (unphosphorylated in the current study, partially phosphorylated at Ser 68 in (74)) supports the hypothesis that the cytoplasmic segment of FXYD1 does not interact properly with the enzyme in the detergent-solubilized system.

4.4 The Lipid Environment Surrounding the Complex Affects the