Conformational transitions of the H,K-ATPase studied with sodium ions as surrogates for protons

Following a recent demonstration that H,K-ATPase can active transport Na+ at a low rate (Polvani, C., Sachs, G., and Blostein, R. (1989) J. Biol. Chem. 264, 17854-17859), we have looked for and found effects of Na+ ions on the conformational state of gastric H,K-ATPase labeled with fluorescein isothiocyanate. Na+ ions reverse the K(+)-induced quench of the fluorescein fluorescence and somewhat enhance fluorescence in the absence of K+ ions. Equilibrium titrations of the cation effects show that Na+ and K+ ions are strictly competitive with apparent dissociation constants of KNa+ = 62 mM (n = 2) and KK+ = 6.6 mM (n = 2). The observations demonstrate that Na+ ions bind to and stabilize the high fluorescence E1 form of the protein while K+ ions stabilize the low fluorescence E2 form. Elevation of pH from 6.4 to 8.0 increased the apparent affinity of the Na+ ions from approximately 62 to 10.2 mM, consistent with competition between protons and Na+. The action of Na+ to stabilize the E1 form was used to measure the rate of the E2K----E1Na transition with a stopped-flow fluorimeter. The rate at pH 6.4 and 20 degrees C is 18.1 s-1. In addition the rate of the reverse conformational transition E1K----E2K has been measured at several K+ concentrations. From the hyperbolic dependence on K+ concentration a maximal rate of 211 +/- 32 s-1 and intrinsic K+ dissociation constant on E1 of 64.6 +/- 3.3 mM have been estimated. The kinetic and equilibrium data are self-consistent and thus support the proposed action of Na+ and K+ ions. Compared with Na,K-ATPase, the H,K-ATPase exhibits a lower affinity for Na+ on E1 and a much faster rate of the E2K----E1Na transition, but a similar affinity for K+ ions on E1 and rate of the transition E1K----E2K. The significance of the similarities and differences in cation specificity and rates of conformational changes of Na,K- and H,K-ATPases is discussed.     

Crystallization of the gastric H,K-ATPase

Crystalline arrays of the gastric H,K-ATPase were obtained in membrane preparations from hog and rabbit gastric mucosa. The lattice was formed rapidly in a medium containing K+, vanadate, Mg2+, and dimethyl sulfoxide at pH 6.0-6.9 in imidazole buffer from 4 to 22 degrees C. The crystal lattice exhibited P2 symmetry, and the unit cell dimension (a = 5.6, b = 11, and c = 10 nm) could accommodate 2 polypeptides of mass 116-129 kDa. In addition, the isolated preparation contained previously undescribed long cylindrical structures 16 nm thick. These structures consisted of a central core 6-7 nm wide from which particles spaced 5.5 nm apart protruded symmetrically.     

Structural aspects of the gastric H,K-ATPase

The gastric H,K-ATPase is an alpha,beta heterodimer. The large catalytic subunit is composed, in the case of the hog enzyme, of 1033 amino acids, whereas the beta subunit is composed of about 291 amino acids and is heavily glycosylated. The membrane topology of the alpha subunit is difficult to predict using hydropathy analysis. Tryptic hydrolysis of intact, inside out vesicles followed by cysteine labelling with fluorescein-5-maleimide provided experimental evidence for an 8 membrane spanning model for the alpha subunit, between residues 104 and 162 (M1/M2), 291 and 358 (M3/M4), 776 and 835 (M5/M6), and 853 and 946 (M7/M8). No evidence was found for a pair of segments (M9/M10) towards the C terminal end of the molecule, contrary to predictions for the Na,K- and Ca-ATPases. Iodination of intact vesicles followed by carboxypeptidase Y cleavage of the C terminal tyrosines showed that the C terminal end of the alpha subunit was cytoplasmic. The epitope for antibody 146 was extracytoplasmic and located between residues 871 to 874 between M7/M8. The binding site of the K competitive imidazo-pyridine, SCH28080, was to the extracytoplasmic loop between M1 and M2, whereas the binding of the covalent SH reagent generated from acid activation of omeprazole in acid transporting vesicles was to 2 cysteines at positions 813 (or 822) and 892 predicted to be in the extracytoplasmic loops connecting M5/M6 and M7/M8, respectively. The beta subunit was only hydrolysed in broken vesicles. A fragment beginning at position 236 was liberated under these conditions only in the presence of reducing agents, showing that cysteine 210 and 263 were disulfide linked. It seems that this subunit has only a single membrane spanning segment as predicted by hydrophobicity. Binding of either SCH28080 or omeprazole to the extracytoplasmic face of the enzyme affected cytoplasmic conformational changes, showing that there was transmembranal transmission of changes of shape of the protein.     

Site-directed antibodies as topographical probes of the gastric H,K-ATPase alpha-subunit.

Gastric acid is secreted by an ATP-driven H+ and K+ exchanger (H,K-ATPase), an integral apical membrane protein of parietal cells. Although the primary structure of the enzyme is known, its higher order structure is uncertain. In order to acquire topographical probes of native, microsomal H,K-ATPase, synthetic peptides corresponding to the 17 amino-terminal (N-peptide) and 16 carboxyl-terminal (C-peptide) residues of pig gastric H,K-ATPase alpha-subunit were coupled to keyhole limpet hemocyanin (KLH). Rabbits were immunized with peptide-KLH conjugates and their sera were tested for specificity by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and immunocytochemistry. All sera showed high ELISA reactivities with synthetic peptides, peptide-BSA conjugates, and microsomal H,K-ATPase adsorbed to microtiter wells (some titers greater than 1:10(4)). Immunoblots of H,K-ATPase resolved by SDS-PAGE showed both N-peptide and C-peptide antibodies reacting with a single 94 kDa band. All sera selectively stained parietal cells in pig gastric mucosal sections. Preimmune sera gave negative or weak signals in all assays. In competition ELISAs, N-peptide antibodies, but not C-peptide antibodies, were displaced from the corresponding bound synthetic peptides by added microsomal H,K-ATPase. One of the N-peptide antibodies inhibited H,K-ATPase activity by more than 50%; binding of this antibody was decreased when ATP or K+ were bound to the enzyme. These results indicate a cytoplasmically-oriented alpha-subunit N-terminus which may participate conformationally in the H,K-ATPase catalytic cycle, and suggest that antibodies against synthetic H,K-ATPase peptides are potentially useful probes of native microsomal H,K-ATPase topography.     

Electrogenic partial reactions of the gastric H,K-ATPase

The fluorescent styryl dye RH421 was used to identify and investigate electrogenic reaction steps of the H,K-ATPase pump cycle. Equilibrium titration experiments were performed with membrane vesicles isolated from hog gastric mucosa, and cytoplasmic and luminal binding of K(+) and H(+) ions was studied. It was found that the binding and release steps of both ion species in both principal conformations of the ion pump, E(1) and P-E(2), are electrogenic, whereas the conformation transitions do not contribute significantly to a charge movement within the membrane dielectric. This behavior is in agreement with the transport mechanism found for the Na,K-ATPase and the sarcoplasmic reticulum Ca-ATPase. The data were analyzed on the basis of the Post-Albers reaction cycle. For proton binding, two pK values were found in both conformations: 6.7 and 相似文献   

Solubilization and reconstitution of the gastric H,K-ATPase

Proteoliposomes containing the hog gastric H+,K+-ATPase were prepared from cholate and n-octyl glucoside extracts of native microsomes. Experiments were presented which show reconstitution-dependent selective purification of a 94-kDa peptide capable of Rb+/Rb+ exchange and active H+ transport. The absence of selective enrichment of residual protein contamination in this material suggests but does not prove that those transport reactions are attributable only to the 94-kDa peptide. Transport demonstrated inhibitor sensitivity and cation specificity comparable to the microsomal gastric ATPase. In K2SO4 media the H+ transport reaction was protonophore insensitive and correlated with MgATP-dependent 86Rb+ extrusion. This and other evidence suggested that active transport occurs via electroneutral H+in for K+out exchange. 86Rb+ exchange (uptake) in the proteoliposomes demonstrated both saturable and nonsaturable components. At a K0.5 = 1.5 mM, saturable 86Rb+ uptake accounted for about 90% of Rb+ influx. The vanadate-sensitive cation exchange indicated that the ATPase was reconstituted asymmetrically into the proteoliposomes (70% cis-/30% trans-vanadate site). 86Rb+ exchange was inhibited by ATP and stimulated about 2-fold by low Mg2+ and 5 mM phosphate. These ligand effects and the demonstration of comparable rates of passive exchange and active Rb+ efflux suggest that passive K+ exchange is not severely limited by a K+-occluded enzyme form in the H,K-ATPase. A model compatible with this hypothesis is suggested.     

Cation selectivity of gastric H,K-ATPase and Na,K-ATPase chimeras.

Chimeras of the catalytic subunits of the gastric H,K-ATPase and Na, K-ATPase were constructed and expressed in LLC-PK1 cells. The chimeras included the following: (i) a control, H85N (the first 85 residues comprising the cytoplasmic N terminus of Na,K-ATPase replaced by the analogous region of H,K-ATPase); (ii) H85N/H356-519N (the N-terminal half of the cytoplasmic M4-M5 loop also replaced); and (iii) H519N (the entire front half replaced). The latter two replacements confer a decrease in apparent affinity for extracellular K+. The 356-519 domain and, to a greater extent, the H519N replacement confer increased apparent selectivity for protons relative to Na+ at cytoplasmic sites as shown by the persistence of K+ influx when the proton concentration is increased and the Na+ concentration decreased. The pH and K+ dependence of ouabain-inhibitable ATPase of membranes derived from the transfected cells indicate that the H519N and, to a lesser extent, the H356-519N substitution decrease the effectiveness of K+ to compete for protons at putative cytoplasmic H+ activation sites. Notable pH-independent behavior of H85N/H356-519N at low Na+ suggests that as pH is decreased, Na+/K+ exchange is replaced largely by (Na+ + H+)/K+ exchange. With H519N, the pH and Na+ dependence of pump and ATPase activities suggest relatively active H+/K+ exchange even at neutral pH. Overall, this study provides evidence for important roles in cation selectivity for both the N-terminal half of the M4-M5 loop and the adjacent transmembrane helice(s).     

Two-dimensional crystals of membrane-bound gastric H,K-ATPase.

Two-dimensional crystallization of membrane-bound H,K-ATPase (EC 3.6.1.36) in vesicle preparations from parietal cells of hog gastric mucosa was induced by an imidazole buffer containing Mg2+ and VO3- ions. A continuous reorganization of the protein molecules started within a few hours by the formation of linear arrays. At later stages confluent two-dimensional crystals were formed. Electron microscopy and image processing showed that these were of a single tetragonal type. The asymmetric unit consisted of one pear-shaped protein domain corresponding to a H,K-ATPase protomer. Through stain-deficient contact regions four adjacent protein units were connected forming a tetrameric structure.     

Site-directed mutagenesis of cation coordinating residues in the gastric H,K-ATPase

Site-mutations were introduced into putative cation binding site 1 of the H,K-ATPase at glu-797, thr-825, and glu-938. The side chain oxygen of each was not essential but the mutations produced different activation and inhibition kinetics. Site mutations thr-825 (ala, leu) and glu-938 (ala, gln) modestly decreased the apparent affinity to K+, while glu-797 (gln) was equivalent to wild type. As expected of competitive inhibition, mutations of thr-825 and glu-938 that decreased the apparent affinity for K+ also increased the apparent affinity for SCH28080. This is consistent with the participation of thr-825 and glu-938 in a cation binding domain. The sidechain geometry, but not the sidechain charge of glu-797, is essential to ATPase function as the site mutant glu-797 (gly) inactivated the H,K-ATPase, while glu-797 (gln) was active but the apparent affinity to SCH 28080 was decreased by four-fold. Lys-793, a unique residue of the H,K-ATPase, was essential for ATPase function. Since this residue is adjacent to site 1, the result suggests that charge pairing between lys-793 and residues at or near this site may be essential to ATPase function.     

Inhibitor and ion binding sites on the gastric H,K-ATPase

The gastric H,K-ATPase catalyzes electroneutral exchange of H(+) for K(+) as a function of enzyme phosphorylation and dephosphorylation during transition between E(1)/E(1)-P (ion site in) and E(2)-P/E(2) (ion site out) conformations. Here we present homology modeling of the H,K-ATPase in the E(2)-P conformation as a means of predicting the interaction of the enzyme with two known classes of specific inhibitors. All known proton pump inhibitors, PPIs, form a disulfide bond with cysteine 813 that is accessible from the luminal surface. This allows allocation of the binding site to a luminal vestibule adjacent to Cys813 enclosed by part of TM4 and the loop between TM5 and TM6. K(+) competitive imidazo-1,2alpha-pyridines also bind to the luminal surface of the E(2)-P conformation, and their binding excludes PPI reaction. This overlap of the binding sites of the two classes of inhibitors combined with the results of site-directed mutagenesis and cysteine cross-linking allowed preliminary assignment of a docking mode for these reversible compounds in a position close to Glu795 that accounts for the detailed structure/activity relationships known for these compounds. The new E(2)-P model is able to assign a possible mechanism for acid secretion by this P(2)-type ATPase. Several ion binding side chains identified in the sr Ca-ATPase by crystallography are conserved in the Na,K- and H,K-ATPases. Poised in the middle of these, the H,K-ATPase substitutes lysine in place of a serine implicated in K(+) binding in the Na,K-ATPase. Molecular models for hydronium binding to E(1) versus E(2)-P predict outward displacement of the hydronium bound between Asp824, Glu820, and Glu795 by the R-NH(3)(+) of Lys791 during the conformational transition from E(1)P and E(2)P. The site for luminal K(+) binding at low pH is proposed to be between carbonyl oxygens in the nonhelical part of the fourth membrane span and carboxyl oxygens of Glu795 and Glu820. This site of K(+) binding is predicted to destabilize hydrogen bonds between these carboxylates and the -NH(3)(+) group of Lys791, allowing the Lys791 side chain to return to its E(1) position.     

cDNA cloning of the beta-subunit of the rat gastric H,K-ATPase

A cDNA encoding the beta-subunit of the rat gastric H,K-ATPase has been identified using oligonucleotide probes based on the amino acid sequences of two peptides from the pig H,K-ATPase beta-subunit (Hall, K., Perez, G., Anderson, D., Gutierrez, C., Munson, K., Hersey, S. J., Kaplan, J. H., and Sachs, G. (1990) Biochemistry 29, 701-706). The nucleotide sequence of the 1.3-kilobase cDNA has been determined and the primary structure of the protein deduced. The protein consists of 294 amino acids and has an Mr of 33,625. The amino acid sequence of the H,K-ATPase beta-subunit is similar to those of the beta 1 (29% identity) and beta 2 (37% identity) subunits of the Na,K-ATPase. Based on the hydropathy profile it seems to have the same transmembrane organization as the Na,K-ATPase beta-subunit, with a single membrane-spanning domain near the amino terminus. Seven potential N-linked glycosylation sites are located in the putative extracellular regions of the protein. Northern blot analyses of poly(A)+ RNAs from 13 tissues demonstrate that the H,K-ATPase beta-subunit mRNA is expressed at high level in stomach and is not expressed in any of the other tissues.     

SDS purification of porcine H,K-ATPase from gastric mucosa

A highly purified membrane fraction of H,K-ATPase was isolated from hog gastric mucosa by using differential centrifugation, sodium dodecyl sulfate (SDS:0.125%) treatment and density-gradient centrifugation. The final fraction showed a major band at 97 kD by SDS-gel electrophoresis. This purified H,K-ATPase sedimented at the interface of a 28-35% sucrose step gradient and displayed a specific activity of 140-170 mumol Pi/h/mg protein and a ratio of K-stimulated ATPase activity to Mg-stimulated ATPase activity of 6.5-8.7. The apparent Km for ATP was 0.154 mM and the Km for K+ was o.6 mM. The enzymatic activity recovered from this purification procedure was K(+)-ionophore-independent. SDS treatment in the presence of 2.5 mM ATP did not change the kinetic properties of the isolated enzyme. Exclusion of ATP during SDS solubilization diminished the enzymatic activity by 90%, indicating that ATP protection is essential for the full recovery of enzymatic activity. In summary, mild SDS solubilization can be used to purify relatively large quantities of active H,K-ATPase to near homogeneity without altering the enzyme's kinetic properties.     

Direct activation of gastric H,K-ATPase by N-terminal protein kinase C phosphorylation. Comparison of the acute regulation mechanisms of H,K-ATPase and Na,K-ATPase

In this study we compared the protein kinase dependent regulation of gastric H,K-ATPase and Na,K-ATPase. The protein kinase A/protein kinase C (PKA/PKC) phosphorylation profile of H,K-ATPase was very similar to the one found in the Na,K-ATPase. PKC phosphorylation was taking place in the N-terminal part of the alpha-subunit with a stoichiometry of approximately 0.6 mol Pi/mole alpha-subunit. PKA phosphorylation was in the C-terminal part and required detergent, as is also found for the Na,K-ATPase. The stoichiometry of PKA-induced phosphorylation was approximately 0.7 mol Pi/mole alpha-subunit. Controlled proteolysis of the N-terminus abolished PKC phosphorylation of native H,K-ATPase. However, after detergent treatment additional C-terminal PKC sites became exposed located at the beginning of the M5M6 hairpin and at the cytoplasmic L89 loop close to the inner face of the plasma membrane. N-terminal PKC phosphorylation of native H,K-ATPase alpha-subunit was found to stimulate the maximal enzyme activity by 40-80% at saturating ATP, depending on pH. Thus, a direct modulation of enzyme activity by PKC phosphorylation could be demonstrated that may be additional to the well-known regulation of acid secretion by recruitment of H,K-ATPase to the apical membranes of the parietal cells. Moreover, a distinct difference in the regulation of H,K-ATPase and Na,K-ATPase is the apparent absence of any small regulatory proteins associated with the H,K-ATPase.     

Phosphorylation of the Na,K-ATPase and the H,K-ATPase

Phosphorylation is a widely used, reversible means of regulating enzymatic activity. Among the important phosphorylation targets are the Na⁺,K⁺- and H⁺,K⁺-ATPases that pump ions against their chemical gradients to uphold ionic concentration differences over the plasma membrane. The two pumps are very homologous, and at least one of the phosphorylation sites is conserved, namely a cAMP activated protein kinase (PKA) site, which is important for regulating pumping activity, either by changing the cellular distribution of the ATPases or by directly altering the kinetic properties as supported by electrophysiological results presented here. We further review the other proposed pump phosphorylations.     

cDNA cloning of the beta-subunit of the human gastric H,K-ATPase.

A full-length cDNA clone encoding the human gastric H,K-ATPase (EC 3.6.1.36)beta-subunit was isolated from a human gastric mucosal lambda gt10 library using oligonucleotide probes which were based on the cDNA sequence from rat and rabbit H,K-ATPase beta-subunits. The insert was 1407 bp in length and encoded a polypeptide of 291 amino acids with a MW = 33,367 Da. It exhibited 84.2%, 85.6% and 81.3% identity to the H,K-ATPase beta-subunits of rabbit, pig and rat, respectively.     

Ouabain-sensitive H,K-ATPase functions as Na,K-ATPase in apical membranes of rat distal colon

Na,K-ATPase activity has been identified in the apical membrane of rat distal colon, whereas ouabain-sensitive and ouabain-insensitive H,K-ATPase activities are localized solely to apical membranes. This study was designed to determine whether apical membrane Na,K-ATPase represented contamination of basolateral membranes or an alternate mode of H,K-ATPase expression. An antibody directed against the H, K-ATPase alpha subunit (HKcalpha) inhibited apical Na,K-ATPase activity by 92% but did not alter basolateral membrane Na,K-ATPase activity. Two distinct H,K-ATPase isoforms exist; one of which, the ouabain-insensitive HKcalpha, has been cloned. Because dietary sodium depletion markedly increases ouabain-insensitive active potassium absorption and HKcalpha mRNA and protein expression, Na, K-ATPase and H,K-ATPase activities and protein expression were determined in apical membranes from control and sodium-depleted rats. Sodium depletion substantially increased ouabain-insensitive H, K-ATPase activity and HKcalpha protein expression by 109-250% but increased ouabain-sensitive Na,K-ATPase and H,K-ATPase activities by only 30% and 42%, respectively. These studies suggest that apical membrane Na,K-ATPase activity is an alternate mode of ouabain-sensitive H,K-ATPase and does not solely represent basolateral membrane contamination.     

Functional consequences of the oligomeric form of the membrane-bound gastric H,K-ATPase

Cross-linking and two-dimensional crystallization studies have suggested that the membrane-bound gastric H,K-ATPase might be a dimeric alpha,beta-heterodimer. Effects of an oligomeric structure on the characteristics of E(1), E(2), and phosphoenzyme conformations were examined by measuring binding stoichiometries of acid-stable phosphorylation (EP) from [gamma-(32)P]ATP or (32)P(i) or of binding of [gamma-(32)P]ATP and of a K(+)-competitive imidazonaphthyridine (INT) inhibitor to an enzyme preparation containing approximately 5 nmol of ATPase/mg of protein. At <10 microM MgATP, E(1)[ATP].Mg.(H(+)):E(2) is formed at a high-affinity site, and is then converted to E(1)P.Mg.(H(+)):E(2) and then to E(2)P.Mg:E(1) with luminal proton extrusion. Maximal acid-stable phosphorylation yielded 2.65 nmol/mg of protein. Luminal K(+)-dependent dephosphorylation returns this conformation to the E(1) form. At high MgATP concentrations (>0.1 mM), the oligomer forms E(2)P.Mg:E(1)[ATP].Mg.(H(+)). The sum of the levels of maximal EP formation and ATP binding was 5.3 nmol/mg. The maximal amount of [(3)H]INT bound was 2.6 nmol/mg in the presence of MgATP, Mg(2+), Mg-P(i), or Mg-vanadate with complete inhibition of activity. K(+) displaced INT only in nigericin-treated vesicles, and thus, INT binds to the luminal surface of the E(2) form. INT-bound enzyme also formed 2.6 nmol of EP/mg at high ATP concentrations by formation of E(2).Mg.(INT)(exo):E(1)[ATP].Mg.(H(+)) which is converted to E(2).Mg.(INT)(exo):E(1)P.Mg.(H(+))(cyto), but this E(1)P form was K(+)-insensitive. Binding of the inhibitor fixes half the oligomer in the E(2) form with full inhibition of activity, while the other half of the oligomer is able to form E(1)P only when the inhibitor is bound. It appears that the catalytic subunits of the oligomer during turnover in intact gastric vesicles are restricted to a reciprocal E(1):E(2) configuration.     

Residues of the fourth transmembrane segments of the Na,K-ATPase and the gastric H,K-ATPase contribute to cation selectivity

We have generated protein chimeras to investigate the role of the fourth transmembrane segments (TM4) of the Na,K- and gastric H, K-ATPases in determining the distinct cation selectivities of these two pumps. Based on a helical wheel analysis, three residues of TM4 of the Na,K-ATPase were changed to their H,K-counterparts. A construct carrying three mutations in TM4 (L319F, N326Y, and T340S) and two control constructs were heterologously expressed in Xenopus laevis oocytes and in the pig kidney epithelial cell line LLC-PK(1). Biochemical ATPase assays demonstrated a large sodium-independent ATPase activity at pH 6.0 for the pump carrying the TM4 substitutions, whereas the control constructs exhibited little or no activity in the absence of sodium. Furthermore, at pH 6.0 the K(1/2)(Na(+)) shifted to 1.5 mM for the TM4 construct compared with 9.4 and 5.9 mM for the controls. In contrast, at pH 7.5 all three constructs had characteristics similar to wild type Na,K-ATPase. Large increases in K(1/2)(K(+)) were observed for the TM4 construct compared with the control constructs both in two-electrode voltage clamp experiments in Xenopus oocytes and in ATPase assays. ATPase assays also revealed a 10-fold shift in vanadate sensitivity for the TM4 construct. Based on these findings, it appears that the three identified TM4 residues play an important role in determining both the specific cation selectivities and the E(1)/E(2) conformational equilibria of the Na,K- and H,K-ATPase.     

Reconstruction of the complete ouabain-binding pocket of Na,K-ATPase in gastric H,K-ATPase by substitution of only seven amino acids

Although cardiac glycosides have been used as drugs for more than 2 centuries and their primary target, the sodium pump (Na,K-ATPase), has already been known for 4 decades, their exact binding site is still elusive. In our efforts to define the molecular basis of digitalis glycosides binding we started from the fact that a closely related enzyme, the gastric H,K-ATPase, does not bind glycosides like ouabain. Previously, we showed that a chimera of these two enzymes, in which only the M3-M4 and M5-M6 hairpins were of Na,K-ATPase, bound ouabain with high affinity (Koenderink, J. B., Hermsen, H. P. H., Swarts, H. G. P., Willems, P. H. G. M., and De Pont, J. J. H. H. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 11209-11214). We also demonstrated that only three amino acids (Phe(783), Thr(797), and Asp(804)) present in the M5-M6 hairpin of Na,K-ATPase were sufficient to confer high affinity ouabain binding to a chimera which contained in addition the M3-M4 hairpin of Na,K-ATPase (Qiu, L. Y., Koenderink, J. B., Swarts, H. G., Willems, P. H., and De Pont, J. J. H. H. M. (2003) J. Biol. Chem. 278, 47240-47244). To further pinpoint the ouabain-binding site here we used a chimera-based loss-of-function strategy and identified four amino acids (Glu(312), Val(314), Ile(315), Gly(319)), all present in M4, as being important for ouabain binding. In a final gain-of-function study we showed that a gastric H,K-ATPase that contained Glu(312), Val(314), Ile(315), Gly(319), Phe(783), Thr(797), and Asp(804) of Na,K-ATPase bound ouabain with the same affinity as the native enzyme. Based on the E(2)P crystal structure of Ca(2+)-ATPase we constructed a homology model for the ouabain-binding site of Na,K-ATPase involving all seven amino acids as well as several earlier postulated amino acids.