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New Molecular Determinants Controlling the Accessibility of Ouabain to Its Binding Site in Human Na,K-ATPase Isoforms

Institute of Pharmacology and Toxicology of the University, Lausanne, Switzerland

Received August 26, 2004; accepted October 22, 2003

	Abstract

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Inhibition of Na,K-ATPase 2 isoforms in the human heart is supposed to be involved in the inotropic effect of cardiac glycosides, whereas inhibition of 1 isoforms may be responsible for their toxic effects. Human Na,K-ATPase 1 and 2 isoforms exhibit a high ouabain affinity but significantly differ in the ouabain association and dissociation rates. To identify the structural determinants that are involved in these differences, we have prepared chimeras between human 1 and 2 isoforms and 2 mutants in which nonconserved amino acids were exchanged with those of the 1 isoform, expressed these constructs in Xenopus laevis oocytes, and measured their ouabain binding kinetics. Our results show that replacement of Met¹¹⁹ and Ser¹²⁴ in the M1–M2 extracellular loop of the 2 isoform by the corresponding Thr¹¹⁹ and Gln¹²⁴ of the 1 isoform shifts both the fast ouabain association and dissociation rates of the 2 isoform to the slow ouabain binding kinetics of the 1 isoform. The amino acids at position 119 and 124 cooperate with the M7–M8 hairpin and are also responsible for the small differences in the ouabain affinity of the ouabain-sensitive 1 and 2 isoforms. Thus, we have identified new structural determinants in the Na,K-ATPase -subunit that are involved in ouabain binding and probably control, in an isoform-specific way, the access and release of ouabain to and from its binding site.

The Na,K-ATPase is composed of a catalytic -subunit with 10 transmembrane segments, which contains cation, ATP, phosphate, and cardiac glycoside binding sites as well as a -subunit that is a type II membrane protein required for the structural and functional maturation of the -subunit (Geering, 2001). Four and three isoforms exist that show a different tissue distribution (for review, see Blanco and Mercer, 1998) and that can produce Na,K-ATPase isozymes with different transport and pharmacological properties (Crambert et al., 2000).

The contribution of different Na,K-ATPase isozymes to the pharmacological or toxic effects of cardiac glycosides is only partially understood. In rodents, the 1 isoform exhibits a nearly 1000-fold lower affinity for cardiac glycosides than 2 or 3 isoforms. Because rat cardiomyocytes express only 1 and 2 isoforms, it has been speculated that inhibition of the sensitive 2 isoform with low doses of cardiac glycoside produces the positive inotropic effect, whereas additional inhibition of 1 isoforms at higher doses leads to the toxic effect (Adams et al., 1982; Maixent et al., 1987). This hypothesis is supported by the recent observation that mouse hearts with genetically reduced levels of 2 isozymes are hypercontractile as a result of increased Ca²⁺ transients, which mimics the inotropic effect of cardiac glycosides. On the other hand, mouse hearts with reduced levels of 1 isoforms are hypocontractile, which resembles cardiac glycoside toxicity (James et al., 1999).

In humans, the situation is complicated by the fact that 1, 2, and 3 isoforms are present in the heart (Wang et al., 1996) and that all human isoforms have a similar high affinity for cardiac glycosides (Crambert et al., 2000; Wang et al., 2001). It has been speculated that an 2 isoform-specific function in the heart could be supported by a compartmentalization of the 2 isoform together with the Na⁺/Ca²⁺ exchanger into microdomains, near the sarco-/endoplasmic reticulum (Juhaszova and Blaustein, 1997). Another argument for a role of human 2 isoforms in the positive inotropic effect of cardiac glycosides is that ouabain binding to 1 isoforms but not to 2 isoforms is efficiently antagonized by K⁺ at physiological concentrations (Crambert et al., 2000).

Compared with 1 isoforms, human 2 isoforms display still another pharmacological difference that could be important for their implication in the inotropic effect of cardiac glycosides. Indeed, 2 isoforms have 5- to 10-fold faster ouabain association and dissociation kinetics than 1 isoforms (Crambert et al., 2000). Because association and dissociation rates vary in parallel, the K_d values are similar for 1 and 2 isoforms. So far, only 1 isoforms have been studied with respect to the localization of the binding site and the molecular determinants of the association and dissociation kinetics of cardiac glycosides. Experimental and modeling data suggest that cardiac glycosides bind to the extracellular surface of the -subunit (Croyle et al., 1997; Middleton et al., 2000; Farr et al., 2002) and that the physical binding site for cardiac glycosides may be formed by the M3/M4 and M5/M6 hairpins (Koenderink et al., 2000). The association rate of ouabain seems to be dependent on the steroid moiety, whereas the dissociation rate depends both on the steroid and the sugar moieties (Yoda, 1974; Kawamura et al., 2001). For 1 isoforms, ouabain association rates are slow and independent of their sensitivity to cardiac glycosides, whereas dissociation rates are low in sensitive isoforms but high in resistant 1 isoforms (Yoda, 1974). In view of these data, it may be predicted that the differences in the ouabain association and dissociation rates in human 1 and 2 isoforms, which have a similar high sensitivity to cardiac glycosides, reflect differences in the accessibility of ouabain to its binding site rather than an alteration of the binding site itself.

In this study, we aimed to characterize the structural determinants that influence association and dissociation rates of cardiac glycosides to Na,K-ATPase and in particular to the `inotropic' 2 isoform. We produced chimeras between human 1 and 2 isoforms or replaced amino acids in the 2 isoform by the corresponding amino acids of the 1 isoform, expressed the mutants in Xenopus laevis oocytes, and determined the association and dissociation rate and the equilibrium binding constants for ouabain. Our results indicate that the M1–M2 and the M7–M8 hairpins contain several specific amino acids that determine the differences in the ouabain binding kinetics in 1 and 2 isoforms and control the access of ouabain to its binding site.

	Materials and Methods

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Mutants and Chimeras. Chimeras of human 1 and 2 isoforms were produced by introducing restriction sites into the 1 cDNA that are present in the 2 cDNA. polymerase chain reaction fragments of 1 cDNA were digested and ligated into the 2 cDNA to replace the corresponding regions (Table 1). Point mutations were introduced into the human Na,K-ATPase 2 isoform, previously cloned into the pSD5 vector (Crambert et al., 2000), by the polymerase chain reaction-based method described by Nelson and Long (1989). The nucleotide sequences of all constructs were confirmed by dideoxy sequencing and cRNAs were prepared by in vitro translation (Melton et al., 1984).

View this table: [in this window] [in a new window]   TABLE 1 Description of chimeras between Na,K-ATPase 1 and 2 isoforms.

Protein Expression in X. laevis Oocytes and Preparation of Microsomes. Stage V–VI oocytes were obtained from X. laevis as described previously (Geering et al., 1996). cRNAs coding for human Na,K-ATPase 1, 2, 1/2 chimeras, or 2 mutants (10 ng/oocyte) were injected into oocytes in the presence of cRNA coding for the human Na,K-ATPase 1-subunits (1 ng/oocyte). Three days after cRNA injection, microsomes were prepared from oocytes as described previously (Geering et al., 1996). Protein concentrations were determined by the method of Lowry et al. (1951).

[³H]Ouabain Binding Kinetics on Oocyte Microsomes. Ouabain binding kinetics were determined as described previously (Crambert et al., 2000). Briefly, oocyte microsomes (final concentration, 11 µg/ml), previously permeabilized by incubation with 0.15 µg of SDS/µg of protein for 25 min at 19°C, were added to a K⁺-free medium containing various [³H]ouabain (Amersham Biosciences, Piscataway, NJ) concentrations (from 3 x 10^–9 to 5 x 10^–8 M) and 4 mM ATP, 4 mM MgCl₂, 100 mM NaCl, and 30 mM imidazole/HCl, pH 7.4. After 2 h at 37°C, aliquots containing 5 µg of protein were removed, rapidly filtered under vacuum on glass-fiber filters (Whatman GF/C), and rinsed three times with 4 ml of an ice-cold buffer containing 100 mM NaCl and 30 mM imidazole/HCl, pH 7.4. Radioactivity bound to filters was counted after addition of 4 ml of scintillation solution (Emulsifier Scintillator Plus; PerkinElmer Life and Anylytical Sciences, Boston, MA). Ouabain binding experiments were performed under the same conditions on microsomes from noninjected oocytes of the same batch to determine ouabain binding to the oocyte, endogenous Na,K-ATPase, and the nonspecific binding. The mean values of these determinations were subtracted from ouabain binding data obtained with microsomes from cRNA-injected oocytes. Nonspecific binding, which was determined by addition of a 1000-fold excess of unlabeled ouabain, was not significantly different in different batches of oocytes and did not exceed 15% of the total binding. Wild-type and all mutant Na,K-ATPase - complexes were expressed in oocytes to a similar level, as reflected by the similar equilibrium [³H]ouabain binding to oocyte microsomes (B_eq; see below and Table 2), and the expression of the exogenous Na,K-ATPase - complexes was 4 to 10 times above that of the endogenous oocyte Na,K-ATPase.

View this table: [in this window] [in a new window]   TABLE 2 Summary of results on the ouabain binding kinetics of chimeras between Na,K-ATPase 1 and 2 isoforms and of point-mutated 2 isoforms

The association and dissociation kinetics of ouabain to wild-type and mutant Na,K-ATPase -subunits were determined as specified in the figure legends. The dissociation rate constant (k_–1) was calculated from the slope of ln B/B_eq versus time plots; B_eq is specific [³H]ouabain binding at equilibrium and B is specific [³H]ouabain binding at several time points after addition of an excess of unlabeled ouabain. The observed first-order association rate constant (k_obs) of ouabain binding was determined as the slope of ln[(B_eq – B)/B_eq] versus time plots. Knowing k_obs, the ouabain concentration ([ouab]) used for association experiments, and the dissociation rate constant (k_–1), we determined the association rate constant (k₊₁) with the equation k_obs = k₊₁[ouab] – k_–1. All curve fittings were done with Kaleidagraph (Abelbeck/Synergy Software, Reading, PA) and unpaired Student's t test with Excel 98 (Microsoft, Redmond, WA) for the Apple Macintosh.

	Results

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The N- and C-Terminal Parts of the Na,K-ATPase -Subunit Contain Determinants for Ouabain Dissociation Kinetics. Fig. 1B shows the characteristic difference in the dissociation rate constant of ouabain between human Na,K-ATPase 1 and 2 isoforms. As described previously (Crambert et al., 2000), the ouabain dissociation rate constant of the 2 isoform was more than 10-fold greater than that of the 1 isoform. To localize the structural determinants that are responsible for these differences, we first prepared two chimeras, 1_M4/2 and 2_M4/1, in which the N-terminal part (up to M4) and the C-terminal part (including the large cytoplasmic loop up to M10) were exchanged between the two isoforms (see Table 1 and Fig. 1A). The ouabain dissociation rate of the 1_M4/2 chimera was seven times slower than that of the 2 isoform and two times faster than that of the 1 isoform (Fig. 1B). The reverse chimera 2_M4/1 exhibited a 2-fold slower ouabain dissociation rate than the 2 isoform and a 5-fold faster rate than that of the 1 isoform. Thus, both the N-terminal part up to M4 and the subsequent C-terminal part comprising M5 to M10 of the -subunit participate in the determination of ouabain dissociation rates with a predominant role of the N-terminal part.

View larger version (45K): [in this window] [in a new window]   Fig. 1. Localization of regions involved in the dissociation rate constants (k–1) of ouabain in human 1 and 2 isoforms and chimeras. A, schematic representation of chimeras between human 1 () and 2 () isoforms. B, ouabain dissociation kinetics of wild-type 1 and 2 isoforms and of 1/2 chimeras. Microsomes were prepared from noninjected oocytes or from oocytes injected with wild-type 1, 2, or 1/2 chimera cRNAs together with 1 cRNA. After incubation of microsomes for 1 h with 5 x 10–8 M [3H]ouabain, unlabeled ouabain (final concentration, 5 x 10–5 M) was added to initiate ouabain dissociation. Ouabain binding was determined after various periods. Ouabain binding caused by endogenous Na,K-ATPase was subtracted from data obtained on microsomes from the cRNA-injected oocytes. Dissociation rate constants (k–1) were calculated as described under Materials and Methods. Data are means ± S.E. of three to nine experiments done in triplicate. 1M4/2 versus 1, p < 0.01; 2/M1–2/2 versus 1M4/2, p = 0.032; 2/1M7–8/2 versus 1M4/2, p = 0.12; 2/1M1–2/2/1M7–8/2 versus 1, p = 0.02.

Regions in the N- and C-Terminal Parts of the Na,K-ATPase -Subunit Involved in the Ouabain Dissociation Kinetics. To define more precisely the region in the N-terminal part of the -subunit that determines ouabain dissociation kinetics, we tested the effect of replacements of the cytoplasmic N terminus (1_Nter/2), of the M1–M2 region (2/1_M1–2/2) or of the M2-M3 region (2/1_M2–3/2) in the 2 isoform by the corresponding regions of the 1 isoform. As shown in Fig. 1B, the presence of the cytosolic N-terminal tail or the M2–M3 region of the 1 isoform did not significantly change the ouabain dissociation rate constant of the 2 isoform. On the other hand, the presence of the M1–M2 region of the 1 isoform decreased the ouabain dissociation rate constant of the 2 isoform near that of the 1_M4/2 chimera, indicating that this region is an important although not unique determinant in the ouabain dissociation kinetics.

To define the contribution of the C-terminal part of the -subunit in the ouabain dissociation rate, we produced a chimera in which the M7–M8 hairpin in the 2 isoform was replaced with that of the 1 isoform. This region was chosen because it has previously been identified to participate in ouabain binding (for references, see Croyle et al., 1997) and because it contains several amino acid differences among human 1 and 2 isoforms (see Fig. 2A). The 2/1_M7–8/2 chimera showed a ouabain dissociation rate similar to that of the 2_M4/1 chimera (Fig. 1B), indicating that the M7–M8 region, in addition to the M1–M2 region, influences ouabain dissociation kinetics. The essential role of these two regions was confirmed by testing a chimera in which both of these regions were replaced in the 2 isoform by the corresponding regions of the 1 isoform. This double chimera (2/1_M1–2/2/1_M7–8/2) exhibited an ouabain dissociation rate very near that of the 1 isoform (Fig. 1B; Table 2).

View larger version (33K): [in this window] [in a new window]   Fig. 2. Identification of amino acids involved in the dissociation rate constants (k–1) of ouabain in human isoforms. A, sequence alignment of human 1 and 2 isoforms. Shown are the regions in the M1–M2 and the M7–M8 hairpins that contain nonconservative amino acid differences in 1 and 2 isoforms (in bold). B to D, ouabain dissociation kinetics of mutant 2 isoforms. Measurement of ouabain dissociation was performed as described in the legend to Fig. 1. B, ouabain dissociation rate constants (k–1) of 2 isoforms mutated in the M1–M2 region. *, p < 0.05, mutants versus the 2/1M1–2/2 chimera. C, ouabain dissociation rate constants (k–1) of 2 isoforms mutated in the M7–8 region. *, p < 0.05 mutants versus the wild-type 2 isoform and the 2/1M7–8/2 chimera. D, ouabain dissociation rate constants (k–1)ofthe 2 isoform mutated in the M1–M2 and M7–M8 regions. *, p < 0.05 mutant versus wild-type 1 isoform. Data are means ± S.E. of two to four experiments done in triplicate.

Amino Acids in the Na,K-ATPase -subunit Involved in the Ouabain Dissociation Rate. Fig. 2A shows the alignment of the human 1 and 2 sequences in the M1–M2 and M7–M8 regions and the nonconservative amino acid differences in the two isoforms. To identify the amino acids in the M1–M2 and M7–M8 regions that are responsible for the 1 phenotype of the double chimera 2/1_M1–2/2/1_M7–8/2 with respect to the ouabain dissociation kinetics, we tested the effect of the replacement in the 2 isoform of several nonconserved amino acids with the corresponding amino acids of the 1 isoform. Replacement of Met¹¹⁹ in the M1–M2 region of the 2 isoform with the corresponding Thr of the 1 isoform (2M119T) produced a 3-fold reduction in the ouabain dissociation rate constant compared with that of the wild-type 2 isoform but it was still higher than that of the 2/1_M1–2/2 chimera (Fig. 2B; Table 2). Simultaneous replacements in the 2 isoform of Met¹¹⁹ and Gly¹¹⁴ or Ala¹³⁴ with the corresponding amino acid of the 1 isoform did not further decrease the ouabain dissociation rate compared with that of the 2M119T mutant. On the other hand, combination of the M119T mutation with a S124Q mutation (2M119T/S124Q) reduced the ouabain dissociation rate of the 2 isoform 5-fold compared with that of the 2/1_M1–2/2 chimera (Fig. 2B). This result shows that the only two non-conserved amino acids in the first extracellular loop of 1 and 2 isoforms contribute significantly to the differential ouabain dissociation rate of the two isoforms.

The M7–M8 region of the human 1 isoform contained in the 2/1_M7–8/2 chimera shows six nonconservative amino acid differences compared with that of the 2 isoform (Fig. 2A). Several of these nonconserved amino acids in the 2 isoform were replaced alone or in combination with the corresponding amino acids of the 1 isoform, but none of these mutations reduced the ouabain dissociation rate of the 2 isoform to that of the 2/1_M7–8/2 chimera (Fig. 2B; Table 2). Only the double mutant 2S878I/E902Q exhibited a significantly lower ouabain dissociation rate constant than the 2 isoform (Fig. 2B). Moreover, combination of the 2S878I/E902Q mutations in the M7–M8 extracellular loop with the M119T/S124Q mutations in the M1–M2 extracellular loop produced a 2M119T/S124Q/S878I/E902Q mutant with an ouabain dissociation rate that was slightly slower than that of the 2M119T/S124Q mutant, but still significantly different from that of the wild-type 1 isoform (Fig. 2B). Thus, despite the apparent, though minor contribution of the M7–M8 region in the determination of the ouabain dissociation rate of the 1 isoform, we were not able to fully mimic its effect by single or double mutations in the 2 isoform. This indicates that more complex intramolecular interactions are involved in the effect of the M7–M8 region of the -subunit on the ouabain dissociation kinetics.

Regions and Amino Acids in the Human Na,K-ATPase -Subunit Involved in the Ouabain Association Rate. As shown previously (Crambert et al., 2000), the association and dissociation rate constants of ouabain vary in parallel in human 1 and 2 isoforms. Therefore, we investigated whether the regions and amino acids in the -subunit that are involved in the ouabain dissociation process were also important for the ouabain association kinetics. Fig. 3 shows that, similar to previous findings (Crambert et al., 2000), the ouabain association rate constant of the human 2 isoform was about 8-fold greater than that of the 1 isoform. Replacement of the M1–M2 hairpin in the 2 isoform by that of the 1 isoform (2/1_M1–2/2) decreased the ouabain association rate by nearly 4-fold, whereas replacement of the M7–M8 hairpin (2/1_M7–8/2) had only a slight effect (Fig. 3; Table 2). However, when both membrane regions of the 2 isoform were replaced by those of the 1 isoform (2/1_M1–2/2/1_M7–8/2), the ouabain association rate constant was similar to that of the wild-type 1 isoform (Fig. 3). Similar to the ouabain dissociation rate, the ouabain association rate of the 2 isoform was significantly decreased in the 2M119T mutant and even more in the 2M119T/S124Q mutant but was not influenced by mutations of other nonconserved amino acids in the M1–M2 or in the M7–M8 hairpins (see Table 2). However, the 2M119T/S124Q/S878I/E902Q mutant exhibited an ouabain association rate similar to that of the wild-type 1 isoform (Fig. 3). All together, these results indicate that the ouabain association and dissociation kinetics are determined by the same structural regions of the Na,K-ATPase -subunit.

View larger version (15K): [in this window] [in a new window]   Fig. 3. Regions and amino acids involved in the association rate constants (k+1) of ouabain in human isoforms. Microsomes were prepared from noninjected oocytes or from oocytes injected with cRNAs coding for wild-type 1, 2, 1/2 chimeras, or 2 mutants together with 1 cRNA. Ouabain binding was carried out in the presence of 1 x 10–8 M [3H]ouabain and determined after various periods (between 0 and 90 min). Ouabain binding caused by endogenous Na,K-ATPase was subtracted from data obtained on microsomes from the cRNA-injected oocytes. Association rate constants (k+1) were calculated from the observed first-order association rate constant (kobs) and the dissociation rate constant as described under Materials and Methods. Data are means ± S.E. of two to four experiments done in triplicate. 2/M1–2/2 versus 1, p = 0.002; 2/1M7–8/2 versus 2, p = 0.53; 2/1M1–2/2/1M7–8/2 versus 1, p = 0.35; 2M119T/S124Q/S878I/E902Q versus 1, p = 0.068.

Regions In the Human -Subunit That Are Involved in the Ouabain Association and Dissociation Rate Control the Ouabain Affinity of Ouabain-Sensitive Na,K-ATPases. All amino acids that so far have been identified to contribute to ouabain affinity of Na,K-ATPase (Croyle et al., 1997; Qiu et al., 2003) are identical in human 1 and 2 isoforms. It should thus be expected that these two isoforms have an identical ouabain affinity that is determined by the physical ouabain binding site and additional amino acids that influence the ouabain affinity through conformational changes. However, as shown previously (Crambert et al., 2000), although human 1 and 2 isoforms are both ouabain-sensitive, they differ about 4-fold in their affinity for ouabain (Fig. 4). Although the K_d values calculated from the k_–1/k₊₁ ratio (i.e., 2.6 and 4.4 nM for 1 and 2 isoforms, respectively) were lower than the K_d values determined by equilibrium binding (i.e., 5.1 and 17.9 nM for 1 and 2 isoforms, respectively), they varied in parallel between the two isoforms. Here, we show that the same regions that influence the ouabain association and dissociation kinetics also influence the ouabain affinity of the human isoforms. Indeed, the chimera 2/1_M1–2/2 exhibited a K_d value for ouabain that was more than 2-fold lower than that of the wild-type 2 isoform. Replacement of the M7–M8 region did not change the affinity of the 2 isoform for ouabain, but the double chimera had the same ouabain affinity as the 1 isoform (Fig. 4; Table 2).

View larger version (13K): [in this window] [in a new window]   Fig. 4. Ouabain affinity of wild-type, human Na,K-ATPase 1 and 2 isoforms, 1/2 chimeras, and 2 mutants. Ouabain binding experiments at equilibrium with various concentrations of [3H]ouabain were performed as described under Materials and Methods. The equilibrium dissociation constants (Kd) were calculated from Scatchard plots. Data are means of two to three experiments done in triplicate.

	Discussion

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By using random mutagenesis (for references, see Croyle et al., 1997) or chimeras between Na,K- and H,K-ATPase (Qiu et al., 2003), nearly 20 amino acids have been identified that influence the sensitivity of Na,K-ATPase to ouabain. These amino acids are located in several transmembrane segments and extracellular loops of the Na,K-ATPase -subunit and are hypothesized to comprise the physical binding site or to affect indirectly ouabain binding by conformational changes. In this study, we have investigated the structural determinants that are responsible for the isoform-specific differences in the ouabain binding kinetics and could identify new amino acids that are involved in ouabain binding and play a role in the access and release of ouabain to and from its binding site.

Results from previous kinetic studies of ouabain binding, which were performed mainly on Na,K-ATPase 1 isozymes, have suggested that the rate of association of ouabain to enzymes from different sources, both sensitive or insensitive, is similar and that the ouabain sensitivity is mainly determined by differences in the dissociation rate (Yoda, 1974; Akera and Brody, 1977). Our observation that the human 2 isozyme exhibits 5- to 10-fold faster ouabain dissociation and association rates than the 1 isoform, despite their similar high ouabain sensitivity, indicates that this prediction is not valid for isoforms other than 1 isoforms.

The amino acids that have previously been identified to be implicated in the ouabain sensitivity of Na,K-ATPase (Croyle et al., 1997; Qiu et al., 2003) are identical in human 1 and 2 isoforms. This points to a similar structure of the ouabain binding pocket in the two isoforms, which determines their similar high ouabain affinity. However, in the present study, we have identified 2 new amino acid positions in the M1–M2 hairpin that differ from those previously characterized and that are major determinants of the fast and slow ouabain binding kinetics of 2 and 1 isoforms, respectively. Replacement of the 2 amino acids at these positions in the 2 isoform by the corresponding amino acids of the 1 isoform decreases both the association and the dissociation rates of ouabain near those observed in the 1 isoform. Two other amino acids with a minor effect on ouabain binding kinetics were also identified in the M7–M8 extracellular loop. Although the precise mechanism is not known, our results suggest that the two extracellular loops cooperate in a synergistic way to control the accessibility of ouabain to its binding site.

Significantly, the amino acids identified in the 1 (Thr¹¹⁹, Gln¹²⁴, Ile⁸⁷⁸, and Gln⁹⁰²) and 2 (Met¹¹⁹, Ser¹²⁴, Ser⁸⁷⁸, and Glu⁹⁰²) isoforms that determine the slow and the fast ouabain binding kinetics, respectively, of the two isozymes, are conserved in all known mammalian 1 and 2 isoforms. The only exception is the highly ouabain-resistant rat 1, which bears a proline residue at the position of Gln¹²⁴ in the human 1 isoform and a phenylalanine residue at the position of Ile⁸⁷⁸. Interestingly, human 3 isoforms possess Thr¹¹⁹, characteristic of 1 isoforms, and Ser¹²⁴, characteristic of 2 isoforms. This may reflect their ouabain association and dissociation rates, which are intermediate between those of 1 and 2 isoforms (Crambert et al., 2000) and are similar to those of the 2M119T mutant (this study). Besides our results on human Na,K-ATPase isozymes (Crambert et al., 2000), few reports exist on the ouabain binding kinetics of isoforms other than 1. However, the ouabain dissociation rate constants reported for putative 2 isoforms in rat heart (Noel and Godfraind, 1984) or vas deferens (Noel et al., 1998) preparations or for rat 2 or 3 isoforms in transfected cells (O'Brien et al., 1994) are in good agreement with our values reported for human 2 and 3 isoforms (Crambert et al., 2000; this study), and are higher than the values reported for various 1 isoforms (Erdmann and Schoner, 1973; Schultheis et al., 1993). These data support the idea that the differences in the ouabain association and dissociation kinetics between 1 and 2 isoforms is a general phenomenon and not restricted to human Na,K-ATPase isozymes.

At present, how the particular amino acids at positions 119 and 124 in the 1 and 2 isoform mediate the slow and fast ouabain kinetics, respectively, is not known. It has been postulated that the interaction of cardiac glycosides with the Na,K-ATPase occurs in at least two steps: an initial rapidly reversible binding step, followed by a conformational change that permits the formation of a more stable ouabain-enzyme complex (Yoda, 1974). In this model, differences in both the association and dissociation rates in the absence of a significant difference in the apparent ouabain affinity, as observed for 1 and 2 isoforms, could be explained by a difference in the flexibility or the accessibility of the ouabain binding site in the two isoforms. Accordingly, in contrast to the 2 isoform, in the 1 isoform, the amino acids at positions 119 and 124 may induce a conformational state of the enzyme that decreases the flexibility or accessibility of the ouabain binding site. The 1 and 2 isoforms may have a different conformation in the ouabain binding state E2P or produce a different conformation after the initial binding step of ouabain. An even more appealing hypothesis is that in the 1 isoform, but not in the 2 isoform, a gate-like structure exists, formed by Thr¹¹⁹, Gln¹²⁴, Ile⁸⁷⁸, and Gln⁹⁰², that impedes ouabain association and dissociation. This concept could best explain our results that replacements of amino acids 119 and 124 in 2 isoforms with the corresponding amino acids of 1 isoforms changes the `on' and `off' rates of ouabain in parallel and that intramolecular interactions between the amino acids in the M1–M2 extracellular loop and the M7–M8 hairpin are involved in the control of the ouabain binding kinetics. It may be speculated that closure or opening of this putative gate-like structure could be mediated by conformational changes during pump cycling. We have indeed shown previously that binding of ouabain to the human 1 isoform is mainly restricted to the phosphorylated E2 conformation, whereas the 2 isoform can also bind ouabain in a nonphosphorylated K⁺-occluded form of the enzyme (Crambert et al., 2000). We may speculate that the gate-like structure for ouabain in the 1 isoform is closed in all but the E2P conformation, whereas in the 2 isoform, this gate-like structure is less efficient and permits an access to the binding site also in the nonphosphorylated state of the Na,K-ATPase. In the 1 isoform, the presence of a threonine and a glutamine at positions 119 and 124, respectively, may promote strong interactions (hydrogen bonds for threonine and electrostatic interactions for glutamine) with other parts of the -subunit that stabilize a closed state of the gate-like structure for ouabain. In addition to interactions with the M7–M8 extracellular loop, interactions of the M1–M2 loop with the M3–M4 extracellular loop could be imagined in view of the presence in the 1 isoform (but not in the 2 isoform) of amino acids bearing hydroxyl groups (Tyr³¹³ and Thr/Ser³¹⁴) or a negative charge (Glu³¹⁰). Considering the disposition of the transmembrane domains of the Na,K-ATPase -subunit predicted from the Ca-ATPase structure (Toyoshima et al., 2000), an interaction between the extracellular loops could partially cover a putative ouabain binding site formed by the M3/M4 and M5/M6 hairpins (Koenderink et al., 2000), and thereby slow the kinetics of ouabain interaction. Unfortunately, because of large differences in the sequences of the extracellular loops between Na,K-ATPase and Ca-ATPase, the model of the structure of the Ca-ATPase does not permit structural and movement predictions of the extracellular domain of the Na,K-ATPase to verify this hypothesis. Nevertheless, it is interesting to note that results of a recent study (Reuter et al., 2003), based on a normal mode analysis of the Ca-ATPase, are compatible with the idea of gate-like structures in P-type ATPases. The results predict that movements of the membrane helices during the calcium transport cycle, `twist open' the luminal side of the protein because of large rearrangements of the extracellular loops (Reuter et al., 2003).

Still another hypothesis could be considered to explain the differences in ouabain binding kinetics in isoforms. It is most likely that the initial step in ouabain binding to the -subunit is mediated by the D-ring of the steroid part and the lactone moiety (Forbush, 1983). This initial binding event opens, in a second, slower step, a binding site for the sugar moiety. This model, which implicates `sugar-docking' to stabilize the interaction between cardiac glycosides and the -subunit, could be fitted to the ouabain binding kinetics of 1 and 2 isoforms. Possibly, the slow sugar-docking step of ouabain binding occurs only in the 1 isoform and is responsible for the slow ouabain association and dissociation rates. On the other hand, in 2 isoforms, sugar docking may not be possible because of the specific amino acid composition of the M1–M2 extracellular loop, and this lack of sugar binding may lead to the rapid ouabain association and dissociation rates.

In conclusion, we have identified new amino acids in the Na,K-ATPase that differentially control discrete steps in the ouabain binding to 1 and 2 isoforms. These findings, which explain the isoform-specific differences in ouabain binding kinetics, may be of importance for the development of new drugs that are able to discriminate between the `inotropic' 2 and the `toxic' 1 isoform of Na,K-ATPase.

	Acknowledgements

 
We thank Jean-Daniel Horisberger for helpful discussions

	Footnotes

 
This work was supported by Swiss National Science Foundation grant 31-64793.01 (to K.G.).

Address correspondence to: Käthi Geering, Institute of Pharmacology et Toxicology of the University, rue du Bugnon 27, CH-1005 Lausanne, Switzerland. E-mail: kaethi.geering{at}ipharm.unil.ch

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