Ion-binding Affinities in the E 1 Conformation

Sodium-titration experiments in the E1 conformation have been performed by adding small aliquots of NaCl up to 100 mM (Fig. 47). In the presence of FXYD1, a ~ 20-30%

higher Na⁺-binding affinity has been detected in all the preparations investigated in the cur-rent study. In Fig. 47, obtained from the average of four identical experiments conducted with one preparation for both the enzyme with and without FXYD1, the half-saturating Na⁺ concentration, K1/2, is 5.8 ± 0.4 mM for α1/His10-β1 and 4.4 ± 0.2 mM for α1/His10 -β1/FXYD1. The Hill coefficient, n, is the same for both preparations, 1.6 ± 0.1, demonstrat-ing that FXYD1 does not affect the cooperativity of Na⁺-binding. The maximum fluores-cence change is ~ 25% larger in the presence of FXYD1.

Figure 47. Sodium-titration experiments in the E1 conformation with α1/His10-β1 (black) and α1/His10 -β1/FXYD1(red).

Potassium-titration experiments in the E1 conformation are shown in Fig. 48. Small ali-quots of KCl have been added up to 10 mM. The curves are obatiend from the average of six identical experiments for one α₁/His₁₀-β₁ preparation and three for one α₁/His₁₀-β₁/FXYD1.

For both preparations, almost the same K⁺-binding affinity is detected. K_1/2 has been deter-mined to be 0.10 ± 0.02 mM for α1/His10-β1 and 0.08 ± 0.01 mM for α1/His10-β1/FXYD1.

Because of the low fluorescence change in the case of α1/His10-β1, ΔFmax < 5%, the error of the fitted result is large, despite the average of several identical experiments. Considering this, the small difference detected needs to be accounted as not significant. The Hill coeffi-cients, n, are comparable, 0.57 ± 0.06 (-FXYD1) and 0.55 ± 0.04 (+FXYD1), respectively.

The maximum fluorescence change is larger of a factor of 2 in the presence of FXYD1.

Figure 48. Potassium-titration experiments in the E1 conformation with α1/His10-β1 (black) and α1/His10-β1/FXYD1(red).

The Rb⁺-binding affinity in the E1 conformation has also been evaluated (Fig. 49). The titrations have been performed by adding RbCl up to 10 mM. The results represent the aver-age of four identical experiments for one α₁/His₁₀-β₁ preparation and three for one α₁/His₁₀ -β1/FXYD1.

Figure 49. Rubidium-titration experiments in the E1 conformation with α1/His10-β1 (black) and α1/His10-β1/FXYD1(red).

For both α₁/His₁₀-β₁ and α₁/His₁₀-β₁/FXYD1, K_1/2 is 0.07 ± 0.02 mM, with a Hill coeffi-cient of 0.65 ± 0.06. Again, the maximum fluorescence change is larger in the presence of FXYD1.

The difference detected in the half-saturating concentrations of Na⁺ ions, but not of K⁺ and Rb⁺ ions, has stimulated further investigation of the effect of FXYD on the Na⁺-binding affinity. To obtain experimental conditions closer to the physiological situation in cardiac myocytes, the Na⁺-binding affinity in the E₁ conformation has been investigated by sodium-titration experiments in the presence of 1 mM free Mg²⁺ and 1.5 µM free Ca²⁺ ions as described in 2.4.1a (Fig. 50). Considering the presence of EDTA in the Buffer used to perform the experiments, the amounts of MgCl₂ and CaCl₂ necessary to obtain the desired concentrations of free Mg²⁺ and Ca²⁺ ions have been calculated with the program WINMAXC32 2.51. As explained in 2.4.1a, Mg²⁺ and Ca²⁺ ions are known to compete with Na⁺ ions for a binding site on the cytoplasmic domain of the protein, in the loop between M6 and M7 (146). Occupation of this site by a Mg²⁺ or Ca²⁺ ion is assumed to impede Na⁺-entrance at the cytoplasmic side (146), thus affecting the Na⁺-binding affinity in the E1 conformation. Indeed, the half-saturating Na⁺ concentrations detected in the conditions of these experiments are smaller than those evaluated in Fig. 47, due to the lower total concentration of divalent cations (~ 1 mM in Fig. 50 versus 5 mM in Fig. 47).

However, the effect of FXYD1 on the Na⁺-binding affinity is similar, independently of the presence of calcium. With and without 1.5 µM free Ca²⁺ ions, the K_1/2 is 4.1 ± 0.3 mM and 4.0 ± 0.3 mM in the absence of FXYD1, while it is 2.8 ± 0.1 mM and 2.7 ± 0.2 mM in the presence of FXYD1. The curves are obtained from the average of three identical experiments in the different conditions conducetd with one preparation for both the enyzme with and without FXYD1.

Figure 50. Sodium-titration experiments in the E1 conformation in the presence of 1 mM free Mg²⁺

and 1.5 µM free Ca²⁺ions with α1/His10-β1 (black) and α1/His10-β1/FXYD1(red). The experiments have been repeated in the absence of calcium (dashed lines).

A further experimental parameter that has been investigated is the lipid composition of the lipid annulus surrounding the detergent-solubilized proteins. The Na⁺-binding affinity in the E1 conformation has been evaluated for a preparation obtained adding a doubled amount of SOPS (0.1 mg/ml SOPS) during the purification of α₁/His₁₀-β₁, described in 2.1.1b (Fig.

51). Higher K1/2 values have been detected (K1/2 = 9.3 ± 0.9 mM for α1/His10-β1 and K1/2 = 6.8 ± 0.6 mM for α1/His10-β1/FXYD1), but a similar difference, despite the increased number of negative surface charges. The results are obtained from the average of four identical experiments for α1/His10-β1 and three for α1/His10-β1/FXYD1.

Figure 51. Sodium-titration experiments in the E1 conformation with the α1/His10-β1 (black) and α1/His10-β1/FXYD1(red) prepared with a doubled amount of SOPS.

The effect of the excess of FXYD1 during the incubation with α₁/His₁₀-β₁ bound to BD-Talon beads in the in vitro reconstitution procedure has been investigated to ensure that the experiments in the presence of FXYD1 are performed under saturating FXYD1 binding. The α1/His₁₀-β₁ isozyme bound to BD-Talon beads has been incubated with molar excesses of FXYD1 up to 10-fold, and the Na⁺-binding affinity has been studied as a crucial parameter that indicates the α1/His10-β1/FXYD1 complex formation (Fig. 52). At least three experi-ments for each α1/His10-β1/FXYD1 preparation have been averaged to obtain the K1/2 values reported in Fig. 52. A maximum effect is found above a 5-fold molar excess of FXYD1.

When the FXYD1-dependent half-saturating Na⁺ concentration is fitted with the Hill func-tion, it is found that about 50% of the Na,K-ATPase molecules are reconstituted with FXYD1 at a FXYD1 molar excess of 1.5 ± 0.3.

Figure 52. Half-saturating Na⁺ concentrations, K1/2, of the α1/His10-β1/FXYD1 complex plotted against the excess of FXYD1 used during the in vitro reconstitution.