Conversion of the Low Affinity Ouabain-binding Site of Non-gastric H,K-ATPase into a High Affinity Binding Site by Substitution of Only Five Amino Acids*

P-type ATPases of the IIC subfamily exhibit large differences in sensitivity toward ouabain. This allows a strategy in which ouabain-insensitive members of this subfamily are used as template for mutational elucidation of the ouabain-binding site. With this strategy, we recently identified seven amino acids in Na,K-ATPase that conferred high affinity ouabain binding to gastric H,K-ATPase (Qiu, L. Y., Krieger, E., Schaftenaar, G., Swarts, H. G. P., Willems, P. H. G. M., De Pont, J. J. H. H. M., and Koenderink, J. B. (2005) J. Biol. Chem. 280, 32349–32355). Because important, but identical, amino acids were not recognized in that study, here we used the non-gastric H,K-ATPase, which is rather ouabain-insensitive, as template. The catalytic subunit of this enzyme, in which several amino acids from Na,K-ATPase were incorporated, was expressed with the Na,K-ATPase β1 subunit in Xenopus laevis oocytes. A chimera containing 14 amino acids, located in M4, M5, and M6, which are unique to Na,K-ATPase, displayed high affinity ouabain binding. Four of these residues, all located in M5, appeared dispensable for high affinity binding. Individual mutation of the remaining 10 residues to their non-gastric H,K-ATPase counterparts yielded five amino acids (Glu312,Gly319, Pro778, Leu795, and Cys802) whose mutation resulted in a loss of ouabain binding. In a final gain-of-function experiment, we introduced these five amino acids in different combinations in non-gastric H,K-ATPase and demonstrated that all five were essential for high affinity ouabain binding. The non-gastric H,K-ATPase with these five mutations had a similar apparent affinity for ouabain as the wild type Na,K-ATPase and showed a 2000 times increased affinity for ouabain in the \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NH}_{4}^{+}\) \end{document}-stimulated ATPase activity in membranes of transfected Sf9 cells.

and explains why the non-gastric H,K-ATPase has a low affinity for this drug.

EXPERIMENTAL PROCEDURES
Construction of Chimeras and Mutants-The chimeras and mutants were constructed from the rat Na,K-ATPase ␣ 1 -subunit containing the R111Q and D122N mutations (12,13) and the rat non-gastric H,K-ATPase ␣ 2 -subunit (10). The rat Na,K-ATPase ␣ 1 -and ␤ 1 -subunits were cloned into the pTLN vector as described earlier (17). This vector is suitable for the Xenopus laevis oocyte expression system (18). The cDNA of the rat non-gastric H,K-ATPase ␣ 2 -subunit, a gift of Dr. H. Binder (19), was cloned with AvaII and EcorV in the pTLN vector. Site-directed mutagenesis was used to generate the mutants described in this paper. All introduced mutations were verified by sequencing. For clarity reasons we used the numbering of the pig Na,K-ATPase, also for residues in parts originating from H,K-ATPase.
The generation of the pFD vector containing the ␣ 2 -subunit of rat non-gastric H,K-ATPase and the ␤ 1 -subunit of rat Na,K-ATPase (pFD-HK␣ 2 -NaK␤ 1 ) that is suited for the baculovirus expression system has been reported before (10). The pFD vector containing the non-gastric H,K-ATPase ␣ 2 -subunit with the five mutations (D312E, S319G, A778P, I795L, and F802C) was generated from the similar pTLN vector by cloning the AatII-EcoRI fragment in the pFD-HK␣ 2 -NaK␤ 1 construct.
Expression in X. laevis Oocytes-X. laevis were sacrificed and parts from ovaries were removed. Oocytes were separated by incubation for 2 h in modified Ringer's solution (90 mM NaCl, 2 mM KCl, 2 mM CaCl 2 , 5 mM MOPS, 2 pH 7.4, 30 units/l penicillin and 30 mg/l streptomycin) containing 2 mg/ml collagenase A. Prophase-arrested oocytes of stages V and VI were selected for injection of cRNA. cRNA synthesis was carried out with the SP6-mMessage Machine kit (Ambion, Austin, TX); as a template the MluI-linearized pTLN vector was used. For the expression of the ␣and ␤-subunits, oocytes were injected with 10 and 2 ng of the corresponding cRNAs, respectively. After injection, the oocytes were incubated for 3 days at 18°C in modified Ringer's solution (17).
Preparation of Total Membranes-Oocytes were disrupted by passing them Ϯ 20 times through a standard 200-l Gilson pipette in homogenization buffer (10 l/oocyt; 250 mM sucrose, 0.5 mM EDTA, 5 mM Tris/HCl (pH 7.4), and "Complete Protease Inhibitor" (according to the instructions of the manufacturer, Roche Diagnostics. The yolk granules were removed by centrifugation of the samples (1000 ϫ g, 4°C, 3 min), and the supernatant was collected. This step was repeated twice. Subsequently, the membranes were pelleted by centrifugation (16,000 ϫ g, 4°C, 30 min). Finally, the pellet was resuspended in homogenization buffer (3 l/oocyte), and the samples were stored at Ϫ20°C.
Generation of Recombinant Viruses-The pFD vectors containing the different cDNAs were transformed to competent DH10bac Escherichia coli cells (Invitrogen) harboring the baculovirus genome (bacmid) and a transposition helper vector. Upon transposition between the Tn7 sites in the transfer vector and the bacmid, recombinant bacmids were selected and isolated (20). Subsequently, insect Sf9 cells were transfected with recombinant bacmids using Cellfectin reagent (Invitrogen). After a 3-day incubation period, recombinant baculoviruses were isolated and used to infect Sf9 cells at a multiplicity of infection of 0.1. Four days after infection, the amplified viruses were harvested.
Preparation of Sf9 Membranes-Sf9 cells were grown at 27°C in 100-ml spinner flask cultures as described by Klaassen et al. (21). For production of H,K-ATPase, 1.5⅐10 6 cells⅐ml Ϫ1 were infected at a multiplicity of infection of 1-3 in the presence of 1% (v/v) ethanol (22) and 0.1% (w/v) pluronic F-68 (ICN, Aurora, OH) in Xpress medium (Biowittaker, Walkersville, MD) as described previously (23). After 3 days, Sf9 cells were harvested by centrifugation at 2000 ϫ g for 5 min. The cells were washed once at 0°C with 0.25 M sucrose, 2 mM EDTA, and 25 mM Hepes/Tris (pH 7.0), resuspended in sucrose/EDTA/Tris buffer, and sonicated at 60 watts (Branson Power Co., Denbury, CT) for 30 s at 0°C. After centrifugation for 30 min at 10,000 ϫ g the supernatant was collected and recentrifuged for 60 min at 100,000 ϫ g at 4°C. The pelleted membranes were resuspended in the above-mentioned buffer and stored at Ϫ20°C.
Protein Determination-The protein concentrations were quantified with the modified Lowry method according to Peterson (24) using bovine serum albumin as a standard.
Western Blotting-The total membrane fraction of X. laevis oocytes was solubilized in sample buffer and separated on SDS-PAGE gels containing 10% acrylamide according to Laemmli (25). For immunoblotting, the separated proteins were transferred to Immobilon-P membranes (Milipore, Co., Bedford, MA). The ␣-subunits of non-gastric H,K-ATPase and the various chimeras were detected with the polyclonal antibody C384-M79 that recognizes the Glu 528 -Met 554 region of the rat non-gastric H,K-ATPase ␣ 2 -subunit (10). The primary antibody was detected using an anti-rabbit secondary antibody, which was labeled with peroxidase (Dako Denmark A/S, Glostrup, Denmark). The ouabain-protein complex was collected by filtration over a 0.8-m membrane filter (Schleicher and Schuell, Dassel, Germany). After washing twice with ice-cold water, radioactivity was analyzed by liquid scintillation analysis.
ATPase Assay-The ATPase activity was determined using a radiochemical method (26). For this purpose, 5 g of Sf9 membranes were added to 100 l of medium containing 2 mM Mg-[␥-32 P]ATP, 10 mM NH 4 Cl, 0.8 mM MgCl 2 , 0.1 mM EGTA, 0.2 mM EDTA, 1 mM TrisN 3 , 50 mM Tris/HCl (pH 7.0), and concentrations of ouabain as indicated. After incubation for 30 min at 37°C, the reaction was stopped by adding 500 l 10% (w/v) charcoal in 6% (v/v) trichloroacetic acid, and after 10 min at 0°C the mixture was centrifuged for 10 s at 10,000 ϫ g. To 0.15 ml of the clear supernatant, containing the liberated inorganic phosphate ( 32 P i ), 3 ml of OptiFluor (Canberra Packard, Tilburg, The Netherlands) was added, and the mixture was analyzed by liquid scintillation analysis. In general, blanks were prepared by incubating in the absence of membranes. ATPase activity is presented as the difference in activity between membranes of HK␣ 2 -expressing cells and mock-infected cells.
Calculations-Data are presented as mean values with standard error of the mean. Differences were tested for significance by means of Student's t test. IC 50 values were determined by analyzing the plots using the non-linear curve-fitting program (Hill equation function) of Orgin 6.1 (OriginLab Corp., Northhampton, MA).

RESULTS
Very recently we showed that substitution of seven unique amino acids present in extracellularly located parts of transmembrane segments M4, M5, and M6 of the ␣ 2 -subunit of the rat gastric H,K-ATPase by the corresponding amino acids of the ␣ 1 -subunit of rat Na,K-ATPase (Glu 312 , Val 314 , Ile 315 , Gly 319 , Phe 783 , Thr 797 , and Asp 804 ) results in a high affinity ouabain-binding site in the gastric H,K-ATPase (15). In the present study we use the non-gastric H,K-ATPase that has a low affinity for ouabain to investigate the binding site in more detail.
Since the seven amino acids mentioned above are all located in the M4, M5, and M6 regions that can be reached from the extracellular space, only amino acid residues located in this region were chosen for further investigation. The extracellularly located parts of M4, M5, and M6 contain 14 amino acids that are different between Na,K-ATPase and non-gastric H,K-ATPase (Fig. 1). To identify the amino acids in this region that are important for ouabain binding, the rat non-gastric H,K-ATPase residues were replaced by their rat Na,K-ATPase counterparts. Different combinations of these mutations were made (Table 1), and expressed together with the rat Na,K-ATPase ␤ 1 -subunit in X. laevis oocytes, whereupon ouabain binding was measured. The polyclonal antibody C384-M79 that recognizes the Glu 528 -Met 554 region of the rat non-gastric H,K-ATPase ␣ 2 -subunit (10) was used to determine the expression levels in the total membrane proteins on a Western blot. Fig.  2A shows that the expression level of each of the mutants was rather similar to that of the recombinant non-gastric H,K-ATPase. [ 3 H]Ouabain binding levels were determined in the presence of either 1 mM ATP or 1 mM P i to obtain the phosphorylated intermediate E 2 -P, which is optimal for ouabain binding. We previously found that ouabain binding to Na,K-ATPase results in a higher level when phosphorylation was carried out with P i , whereas phosphorylation of a chimera between Na,K-ATPase and gastric H,K-ATPase (HN34/56) only occurs when phosphorylation is carried out with ATP. Studies with vanadate indicate that the latter chimera is mainly in the E 1 -form, whereas the wild type enzyme in mainly in the E 2 form.
The [ 3 H]ouabain binding levels of mutants 2 (ϭ mutations AϩBϩD) and 5 (ϭ mutations AϩBϩCϩD) (Fig. 2, B and C), as determined with 250 nM ouabain in the presence of either 1 mM ATP or 1 mM P i , were significantly higher than that of the recombinant non-gastric H,K-ATPase wild type (p Ͻ 0.05). This shows that the mutations A, B, and D together are sufficient for gaining the ability to bind ouabain with a high affinity. The [ 3 H]ouabain binding levels of mutants 1, 3, and 4 were rather similar to that of the non-gastric H,K-ATPase wild type (p Ͼ 0.05). This demonstrates that none of the later mutants were able to bind ouabain under these conditions. This means that amino acid residues in group C, located in M5, are apparently not important for high affinity ouabain binding. There was hardly any difference between the results with either ATP or P i , suggesting that the enzyme can be easily converted from the E 1 into the E 2 form and oppositely. As a control, wild type Na,K-ATPase gave higher ouabain binding levels when the binding was carried out in the presence of P i (13). The mutants can apparently easily move between the E 1 and E 2 form, so that the forward or backward reaction gives similar results. Taken together, these findings suggest that maximally 10 amino acid residues that are present in the catalytic subunit of Na,K-ATPase but not in that of non-gastric H,K-ATPase are important for ouabain binding. Five of these amino acids are located in M4, two in M5, and three in M6 of Na,K-ATPase (Fig. 1).
To assess which of these 10 amino acids are important for high affinity ouabain binding, we generated individual mutants of mutant 2, in which each of these ten amino acids was back mutated into the original non-gastric H,K-ATPase residue. Western blotting showed again similar expression levels of the new mutants compared with mutant 2 (Fig.  3A). All mutants could be phosphorylated by ATP (data not shown), which excludes the possibility that the loss of ouabain binding capacity of these mutants is due to the impossibility to form a phosphorylated intermediate. Fig. 3, B and C, show that the [ 3 H]ouabain binding levels of mutants Mut2-E312D, Mut2-G319S, Mut2-P778A, Mut2-L795I, and Mut2-C802F were significantly lower than those of mutant 2, indicating that these five mutants lost their ability to bind ouabain with high affinity. We cannot distinguish whether this loss is due to a change in affinity or in a decrease in the stoichiometry of binding sites. The [ 3 H]ouabain binding levels of mutants Mut2-T309Y, Mut2-V314I, Mut2-T781C, and Mut2-V798I were not significantly different from those of mutant 2 (p Ͼ 0.05). The [ 3 H]ouabain binding level of mutant Mut2-W310V was even significantly higher than that of mutant 2 (p Ͻ 0.05). Also in this case the results in the presence of ATP (Fig. 3B) were rather similar to those in the presence of P i (Fig. 3C). In the latter case significance was reached for all mutants except mut2-E312D, which is probably due to the relatively larger experimental variations. Taken together, these observations indicate that Glu 312 , Gly 319 , Pro 778 , Leu 795 , and Cys 802 are essential for high affinity ouabain binding.
However, these kinds of "loss-of-function" experiments could not exclude the possibility that the loss of ouabain binding capacity of the mutations is due to indirect effects on ouabain binding. Therefore we performed "gain-of-function" experiments by introducing different combinations of the above-mentioned five amino acids into non-gastric H,K-ATPase. Fig. 4A shows similar expression levels of all tested

TABLE 1 Description of the used mutants
The mutants used in this study have the rat non-gastric H,K-ATPase ␣ 2 -subunit as a backbone and contain the ␤ 1 -subunit of rat Na,K-ATPase.

Constructs
Mutations AϩBϩCϩD Ouabain Binding to Non-gastric H,K-ATPase MAY 12, 2006 • VOLUME 281 • NUMBER 19 mutants. None of the mutants showed higher [ 3 H]ouabain binding levels than those of non-gastric H,K-ATPase except the mutant with all five mutations, HK␣ 2 -EGPLC (Fig. 4, B and C). Since the individual mutants all could be phosphorylated, it is unlikely that the lack of ouabain binding of most of these mutants is due to an inability to become phosphorylated. These findings confirm that the combination of Glu 312 , Gly 319 , Pro 778 , Leu 795 , and Cys 802 is essential for high affinity ouabain binding.
To compare the apparent affinity for ouabain of chimera HK␣ 2 -EGPLC and Na,K-ATPase, we performed an ouabain replacement assay (Fig. 5) in the presence of 1 mM P i . The figure shows that the apparent ouabain binding affinity of chimera HK␣ 2 -EGPLC (0.22 Ϯ 0.03 M) is not significantly different from that of Na,K-ATPase (0.19 Ϯ 0.01 M) (p Ͼ 0.05), indicating that chimera HK␣ 2 -EGPLC has gained high affinity ouabain binding.
Finally, we expressed chimera HK␣ 2 -EGPLC and the rat Na,K-ATPase ␤ 1 -subunit in Sf9 cells using the baculovirus expression system. The expressed non-gastric H,K-ATPase EGPLC had an NH 4 ϩ -stimu-lated ATPase activity of 9.8 Ϯ 0.9 mol/mg of protein/h that was about 40% of that of the wild type enzyme (26.8 Ϯ 2.8 mol/mg of protein/h). The phosphorylation level of the chimera was 9.0 Ϯ 0.8 pmol/mg of protein that is also about 40% of that of the wild type enzyme (22.5 Ϯ 2.3 pmol/mg of protein). This means that the mutations had no effect on the turnover number of this enzyme. Next, we determined the effect of ouabain on the NH 4 ϩ -stimulated ATPase activity in membranes isolated from these cells. Fig. 6 shows that the IC 50 for ouabain under these conditions is a factor 2000 lower than that of the same preparation without these mutations. This strongly supports the conclusion of this paper that the presence of these five amino acid residues in non-gastric H,K-ATPase is sufficient for yielding a high affinity ouabain-binding site.

DISCUSSION
This paper shows that the low affinity binding site for ouabain in the rat non-gastric H,K-ATPase can be converted into a high affinity binding site by replacement of only five amino acids by their counterparts present in the catalytic subunit of rat Na,K-ATPase. This result was  Table 1. *, significantly different from non-gastric H,K-ATPase wild type (p Ͻ 0.05).  Table 1. All point mutations were made in mutant 2. *, significantly different from mutant 2 (p Ͻ 0.05).

Ouabain Binding to Non-gastric H,K-ATPase
obtained by a systematic mutational approach. Advantage was taken from our previous study (15) in which we were able to transfer the high affinity binding site for ouabain to the ouabain-insensitive gastric H,K-ATPase by mutation of only seven amino acids, originating from Na,K-ATPase. All these seven amino acids are present in extracellularly located parts of M4, M5, and M6 of Na,K-ATPase.
It is known that ouabain binds to the Na,K-ATPase from the extra-cellular side and that the highest affinity for ouabain is obtained when the enzyme is in the phosphorylated E 2 -P form (27). In the extracellular half of M4, M5, and M6 there are 14 amino acids that are different between non-gastric H,K-ATPase and Na,K-ATPase. Introduction of these 14 amino acid residues from Na,K-ATPase into non-gastric H,K-ATPase resulted in an enzyme with a high affinity for ouabain. These 14 amino acid residues were divided into four groups, A, B, C, and D, containing five, two, four, and three mutated amino acid residues, respectively. We next showed that mutant C, which represents a region with a relatively large species difference, did not alter ouabain binding and that only the 10 amino acid residues originating from the groups A, B, and D might be important for the high affinity ouabain binding. Next, individual mutational analysis revealed that only mutants mut2-E312D, mut2-G319S, mut2-P778A, mut2-L795I, and mut2-C802F partly or completely lost the ability to bind [ 3 H]ouabain. Finally, we introduced Glu 312 , Gly 319 , Pro 778 , Leu 795 , and Cys 802 into the ouabain-insensitive non-gastric H,K-ATPase, resulting in chimera HK␣ 2 -EGPLC that dem-   ϩ -stimulated ATPase activity in the absence of ouabain was for both preparations set at 100% and the activities obtained in the presence of the indicated ouabain concentrations were expressed as percentage of these activities. The 100% value for the nongastric H,K-ATPase was 22.5 Ϯ 2.3 mol/mg of protein/h and for the EGPLC mutant 9.0 Ϯ 0.8 mol/mg of protein/h. E, non gastric H,K-ATPase; F, HK␣ 2 -EGPLC. onstrated high affinity ouabain binding (Fig. 5). The apparent affinity of this chimera is much higher than that of the parent non-gastric H,K-ATPase and the rat Na,K-ATPase, whereas it is similar to that of the recombinant high affinity rat (R111Q and D122N) Na,K-ATPase. In addition, the NH 4 ϩ -stimulated ATPase activity measured in membranes isolated from Sf9 cells in which this chimera had been expressed showed a 2000 times higher affinity for ouabain than the wild type non-gastric H,K-ATPase (Fig. 6). These results revealed that Na,K-ATPase residues Glu 312 , Gly 319 , Pro 778 , Leu 795 , and Cys 802 are important for high affinity ouabain binding. Glu 312 and Gly 319 were also found to be important for high affinity ouabain binding in our previous study in which a high affinity ouabain site was transferred to gastric H,K-ATPase (15). In that paper we presented a docking model (Fig. 7), in which Glu 312 forms a hydrogen bridge with a hydroxyl group of the rhamnose sugar in the ouabain molecule, suggesting that Glu 312 has a direct interaction with ouabain.
Gly 319 is present in the ␣-helix of M4 that has van der Waals interactions with the steroid part of ouabain. It is striking that a Gly is also present in non gastric H,K-ATPase in man, guinea pig and Bufo. In rabbit, however, it is an Ala, and in rat a Ser. Asano et al. (4) showed that the guinea pig non-gastric H,K-ATPase expressed in mammalian cells had a relatively high ouabain sensitivity (IC 50 ϭ 52 M). The 86 Rb uptake in Bufo bladder, which is mediated by non-gastric H,K-ATPase, was about two times more sensitive to ouabain than that of the rat enzyme following expression in X. laevis oocytes (16). Oocytes co-injected with the human non-gastric H,K-ATPase cRNA and that of rabbit gastric H,K-ATPase ␤-subunit showed an 86 Rb ϩ uptake that was rather sensitive to ouabain (K i ϭ 13 M) (2). It is likely that the presence of any side chain on position 319, as present in rat and rabbit has a structurally inhibitory effect on ouabain binding.
Interestingly, we found that when Trp 310 was back mutated into the Val residue in mutant 2, the [ 3 H]ouabain binding level of this mutant significantly increased, suggesting that this mutation leads to an ouabain-binding-favorable enzyme conformation. This observation is consistent with the finding that a monoclonal antibody which recognizes the EYTW 310 LE sequence at the M3-M4 junction of Na,K-ATPase enhances the rate of ouabain binding (28). The Trp 310 can give appar-ently spatial hindrance for ouabain binding. Substitution of this residue in mutant 2 or binding of an antibody to this residue (or its environment) in Na,K-ATPase can remove this spatial hindrance.
The other three amino acids that were found to be important for high affinity ouabain binding Pro 778 , Leu 795 , and Cys 802 have not been described before to be important for ouabain binding in Na,K-ATPase. Pro 778 and Leu 795 are present in both Na,K-ATPase and gastric H,K-ATPase and were therefore not identified by making chimeras between these two enzymes. Ala and Ile are present at the corresponding locations in non-gastric H,K-ATPase. Two of the three newly detected amino acids (Pro 778 and Cys 802 ) are most likely not directly involved in ouabain binding (Fig. 7) but support ouabain binding indirectly. Leu 795 might directly contribute to ouabain binding.
From a structural point of view, Pro is an unusual amino acid in which the side chain is cyclized back to the backbone amide position. The backbone conformation is therefore restricted, which leads to a kinked structure (29). A Pro kink provides a way to make curved helices that could be packed into a funnel-like or cage-like structure (30). Such a packing is important for membrane proteins in which structural interactions between helices can be maximized (31). Because Pro 778 is not included in the proposed ouabain binding pocket (15), it is reasonable to postulate that the dramatic structural difference that occurs when Ala is replaced by Pro will help the enzyme to build up a conformation favorable for ouabain binding.
Leu 795 is located close to the extracellular loop (PLPL 795 GTV) between M5 and M6 (Fig. 7). It is likely to be involved in ouabain binding, since ouabain binds to the enzyme from the extracellular side. In addition, substitution of its neighboring amino acid Leu 793 with Pro has been reported to lead to ouabain resistance. The apparent affinity of the L793P mutant for K ϩ is 2-fold lower than that of wild type Na,K-ATPase pump, indicating that the mechanism of ouabain insensitivity of L793P is due to a perturbation in the region of the enzyme that may include the K ϩ -binding site (32). Moreover, substitution of another neighboring residue, Thr 797 with Val and Ala, was found to result in a 79-and 66-fold increase in the IC 50 values for ouabain, respectively (33). In agreement with this we demonstrated before that Thr 797 is one of the three amino acids in transmembrane hairpin M5-M6 of Na,K-ATPase that play a key FIGURE 7. Model for the ouabain-binding pocket in Na,K-ATPase. The model is an extension of the model presented by Qiu et al. (15). The five amino acid residues that were detected in the present study are indicated by arrows. The backbones of the seven amino acids already found in the model of Qiu et al. (15) are yellow. Nitrogen is blue, oxygen red, and carbon cyan or green (in ouabain). Hydrogen bonds are shown as yellow disks. The surrounding parts of the structure have been hidden for clarity. Images were created with Yasara.
role in ouabain binding (14). Taken together, these findings show that the loop region (PLPL 795 GTV) is clearly important for the interaction between ouabain and Na,K-ATPase. Therefore, replacement of Leu 795 with Ile might modify the loop structure that harbors Leu 793 and Thr 797 in such a way that ouabain cannot bind very well.
We also found that replacement of Cys 802 by a Phe resulted in a loss of ouabain binding. Cys 802 is located in the neighborhood of the cationbinding site and cannot be part of the ouabain-binding site. This mutation gives a large structural change from a hydrophilic residue with side chains containing sulfur atoms to hydrophobic amino acid with an aromatic ring. It is possible that this large structural change affects the enzyme conformation and so changes the ouabain affinity. Surprisingly, a similar change in our previous study with gastric H,K-ATPase had much less effect (14). The direct environment of Cys 802 in gastric H,K-ATPase is different from that of non-gastric H,K-ATPase, which might explain the different result. For instance, in gastric H,K-ATPase a Glu is present on position 804, whereas in non-gastric H,K-ATPase, like in Na,K-ATPase, an Asp is present on this position. We showed before that the presence of Asp 804 , which is part of the cation-binding pocket, is necessary for high affinity ouabain binding (13,14). Therefore, it is likely that Cys 802 may alter the ouabain binding affinity indirectly by changing enzyme-cation interactions.
In summary, we presented evidence that the presence of Glu 312 , Gly 319 , Pro 778 , Leu 795 and Cys 802 in Na,K-ATPase is necessary for obtaining high affinity ouabain binding. Moreover, we demonstrated that only these five amino acids present in the extracellular half of M4, M5, and M6 of Na,K-ATPase are sufficient to confer high affinity ouabain binding to non-gastric H,K-ATPase.