From the Institute of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland
Received for publication, January 28, 2003
, and in revised form, March 7, 2003.
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ABSTRACT |
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INTRODUCTION |
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The intramembranous part of the group IIc P-ATPases is made up of 10 transmembrane segments of the -subunit plus the single transmembrane segment of the associated
-subunit. The
-subunits of the Na,K- and H,K-ATPases contain between 5 and 7 negatively charges residues in the transmembrane segments (this count does not include the charged residues located at the intracellular or extracellular borders of most of the 10 transmembrane segments). Within the transmembrane segments, no positively charged amino acids are present in any of the known sequences of the Na,K-ATPase
-subunit, whereas a single positively charged residue, a highly conserved lysine, is present in all known sequences of the gastric and non-gastric H,K-ATPases (for instance, Lys800 in the Bufo marinus bladder H,K-ATPase
-subunit sequence). A similarly highly conserved serine residue (for instance, Ser775 in the sheep
1-subunit sequence) is present at the corresponding position in all Na,K-ATPase
-subunit sequences. Fig. 1 shows a comparison of the highly conserved sequences of the fifth transmembrane segment of the Na,K- and H, K-ATPases
-subunits.
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Because of the coincidence of electrogenic and non-electrogenic types of activity in the absence and presence, respectively, of a positively charged residue in the middle of the fifth transmembrane segment, we hypothesized that this charged amino acid has a major role in the stoichiometry of cation transport by the group IIc P-ATPases. We tested this hypothesis by studying the electrogenicity of cation transport by the Na,K- and non-gastric H,K-ATPases mutated at this single position.
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EXPERIMENTAL PROCEDURES |
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Expression in Xenopus OocytesOocytes were obtained from Xenopus females anesthetized by exposure to MS222 (2 g/liter; Sandoz, Basel, Switzerland) and prepared as described previously (14). Oocytes were injected with 8 ng of wild-type or mutant bl H,K-ATPase cRNA or with 8 ng of
1 Na,K-ATPase cRNA in combination with 1 ng of B. marinus bladder
-subunit cRNA (15). Expression of wild-type and mutant Na,K- and H,K-ATPases was examined by pulse (24 h)-chase (48 h) experiments using [35S]methionine metabolic labeling. Microsomes were prepared as described previously (16) and loaded onto 513% SDS-polyacrylamide gels. For the Na,K-ATPase mutants, the
-subunit was also immunoprecipitated using anti-Bufo
1-subunit antibody (17) under nondenaturing conditions as described (18), allowing co-immunoprecipitation of the associated
-subunit. The dissociated immune complexes were separated by SDS-PAGE, and labeled proteins were detected by fluorography. Quantification of immunoprecipitated bands was performed with an Amersham Biosciences Ultrascan 2202 laser densitometer.
Electrophysiological MeasurementsThree days after injection, the oocytes were loaded with Na+ by a 2-h exposure to a K+-free solution as described (19). The incubation solution contained 0.2 µM ouabain to inhibit the endogenous Xenopus oocyte Na,K-ATPase; we have shown previously (6, 20) that this concentration of ouabain is sufficient to fully inhibit the endogenous Xenopus oocyte Na,K-ATPase and that the dissociation rate constant of ouabain from the Xenopus oocyte Na,K-pump is slow enough so that inhibition is maintained during the duration of the electrophysiological recording even if no ouabain is included in the measurement solutions. Currents associated with the activity of the putative H,K- and Na,K-ATPases were measured by the two-electrode voltage-clamp technique using a TEV-200 voltage-clamp apparatus (Dagan Corp., Minneapolis, MN). Current signals were filtered at 20 Hz and recorded on a Gould Model 220 paper chart recorder. The intracellular potential was held at 50 mV. Current/voltage curves were obtained by applying series of 500-ms voltage steps ranging from 150 to + 30 mV.
The Na,K-pump function was evaluated in an Na+-rich solution (100 mM sodium gluconate, 0.82 mM MgCl2, 0.41 mM CaCl2,10mM N-methyl-D-glucamine/Hepes, 5 mM BaCl2, and 10 mM tetraethylammonium chloride, pH 7.4) or in an Na+-free solution (a similar solution in which 100 sodium gluconate was replaced with 160 mM sucrose) by activation of the current induced by addition of K+ (10 mM K+ when an Na+-rich solution was used or 5 mM K+ when an Na+-free solution was used) to a previously K+-free solution and by the effect of 2 mM ouabain in the K+-containing solution. In another set of experiments, the kinetics of activation of the current by K+ were studied in Na+-rich and Na+-free solutions by measuring the current induced by increasing concentrations of extracellular K+ (0.3, 1, 3, and 10 mM K+ in Na+-rich solution and 0.02, 0.1, 0.5, and 5 mM K+ in Na+-free solutions) as described previously (19). The maximal K+-activated current (Imax) and the half-activation constant (k1/2) were determined as the best fitting parameters of the Hill equation using a Hill coefficient of 1.6 for the measurements performed in the presence of Na+ and a Hill coefficient of 1.0 for those performed in the absence of extracellular Na+ as described previously (19).
Whereas the K+-induced current is a reliable indicator of the Na,K-pump activity at low K+ concentrations, increasing the extracellular K+ concentration above 10 mM results in sizable and variable inward currents independent of the activity of the Na,K-pump. These currents are probably due to K+ flowing through channels that are insufficiently blocked by Ba2+ and tetraethylammonium; thus, for some mutants of the Na,K-ATPase showing a very low affinity for K+, the electrogenic transport was evaluated as the current sensitive to 2 mM ouabain in the presence of 40 mM extracellular K+ (a solution similar to the Na+-rich solution described above, except that Na+ was partially replaced with K+, yielding 60 mM Na+).
86Rb Uptake Measurements86Rb uptake was performed as previously described (16). Briefly, after loading the oocytes with Na+ (see above), oocytes were transferred to a solution containing 5 mM KCl, 90 mM NaCl, 1 mM CaCl2, 5 mM BaCl2, 1 mM MgCl2, 10 mM Hepes, pH 7.4, 0.2 µM ouabain (to inhibit the endogenous oocyte Na,K-ATPase), and 10 µM bumetanide (to inhibit the uptake of 86Rb by the Na-K-2Cl cotransporter). After addition of 5 µCi/ml 86Rb (Amersham Biosciences), oocytes were incubated for 12 min at room temperature and than washed in a solution containing 90 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, and 10 mM Hepes, pH 7.4. Individual oocytes were then dissolved in 0.5% SDS and counted. In each experiment, a group of 1012 oocytes injected with the -subunit alone was used a control. The transport activity of the expressed wild-type or mutant ATPase was estimated as the 86Rb uptake by an experimental oocyte minus the mean 86Rb uptake measured in the same experiment by the group of oocytes injected with the
-subunit alone. As described above, some mutants had a low affinity for extracellular K+. For these mutants, the Na,K-pump transport function was evaluated as 86Rb uptake sensitive to 2 mM ouabain in 40 mM K+ solution (sodium replacement).
Statistical AnalysisData are presented as means ± S.E. (n = number of observations). Statistical analysis of the data was performed by paired Student's t test when pairs of measurements obtained in the same oocyte were compared or by the unpaired Student's t test when different groups of oocytes were compared.
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RESULTS |
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We chose to coexpress these wild-type and mutant -subunits with the B. marinus bladder isoform of the
-subunit (
bl), which is the amphibian homolog of the mammalian
2-isoform, because the
1/
bl and
bl/
bl dimers are well expressed at a similar level in Xenopus oocytes (8), and it was essential to express the various wild-type and mutant
-subunits with the same
-subunit to be able to attribute the observed differences to the mutations carried by the
-subunit.
Expression of wild-type and mutant Na,K- and H,K-ATPases was first examined using metabolic labeling. Fig. 2A shows that all mutant H,K-ATPase -subunits were expressed at similar levels as the wild-type H,K-ATPase
-subunit. All
-subunits appeared stable after a 48-h chase period, which indicates that they are associated with the
-subunit (16). The same was observed for all of the mutant Na,K-ATPase
-subunits (Fig. 2B). A more detailed analysis by immunoprecipitation (Fig. 2C) revealed that
-subunit mutants S782A and S782K, similar to the wild-type
-subunit, were associated with a fully glycosylated
-subunit after a 48-h chase period, indicating their correct routing toward the plasma membrane. In contrast, the S782E mutant and, to a lesser extent, the S782R mutant remained associated with a core glycosylated
-subunit, indicating their (partial) retention in the endoplasmic reticulum.
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We then evaluated the functional expression of the different mutants of the Na,K- and H,K-ATPases in comparison with wild-type pumps using the 86Rb uptake assay. Fig. 3A shows that, in the presence of 5 mM K+, the K800S and K800A mutants of the H,K-ATPase were functionally expressed at similar levels as the wild-type Na,K- and H,K-ATPases, with rubidium transport values of 4060 pmol/min/oocyte above the background uptake observed in control oocytes injected with
bl alone. The K800R and K800E mutants of the H,K-ATPase had smaller but significant levels of rubidium uptake (
520 pmol/min/oocyte). Both
1 Na,K-ATPase and
bl H,K-ATPase can be inhibited by high concentrations of ouabain (9, 12); and in another series of experiments, we tested the sensitivity of 86Rb uptake to ouabain by comparing the uptake in the presence and absence of 2 mM ouabain. As shown in Fig. 3B, except for the K800E mutant, for which no significant ouabain-sensitive 86Rb uptake could be detected, a large part of the 86Rb uptake by the mutants was sensitive to a high concentration (2 mM) of ouabain, similar to wild-type
bl H,K-ATPase, as expected from the known low sensitivity observed in previous measurements (9, 12).
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No significant rubidium transport could be detected under similar conditions (in the presence of 5 mM K+) in oocytes expressing the various mutants of 1 Na,K-ATPase (Fig. 3A). Only the S782A mutant had a very small but significant 86Rb uptake of 2.6 ± 1.1 pmol/min, whereas the wild-type Na,K-ATPase expressed a transport activity of 45.1 ± 8.6 pmol/min, similar to that of the wild-type H,K-ATPase. A low affinity for extracellular K+ has been observed with mutants of the corresponding position (Ser775) in
1 Na,K-ATPase from other species (21, 22, 23). Assuming a similar effect of homologous mutations in the Bufo
1 Na,K-pump, we studied the transport function of the Ser782 mutants at a higher (40 mM) concentration of K+. Under these conditions (Fig. 3C), significant ouabain-sensitive 86Rb uptake could be observed in the different Na,K-ATPase mutants. The rate of transport was, however, smaller than for the wild-type pump (1545 pmol/min/oocyte for the mutants versus 105 pmol/min/oocyte for the wild type, n = 4348 oocytes in each group). These results indicate that all of the mutants were indeed expressed and were able to perform rubidium uptake, although some of them with a lower affinity and at a reduced level of activity compared with the wild-type Na,K-ATPase.
Electrogenic Transport by the Lys800 Mutants of the H,K-ATPaseThe activity of the wild-type and Lys800 mutant H,K-ATPases was first examined in the presence of 100 mM extracellular Na+ as the current activated by 10 mM K+ and as the current inhibited by 2 mM ouabain in the presence of 10 mM K+ (Fig. 4, A (examples) and C (mean values)). When studied under these conditions, the wild-type Na,K-pump generated outward currents amounting to 120 nA; and as reported previously (8), activation by extracellular K+ results only in small inward currents in oocytes expressing the wild-type Bufo bladder H,K-ATPase. An K+-activated and ouabain-sensitive outward current was observed with the K800A mutant H,K-ATPase and, with a very small amplitude, with the K800E mutant H,K-ATPase, indicating the presence of electrogenic transport activity. The smaller amplitude of the outward current expressed by the K800E mutant can be related to the lower transport activity of this mutant (Fig. 3, A and B) and might be due to a slower turnover rate of the transport cycle or to a lower level of expression of this protein at the cell surface even though the protein is synthesized and appears stable (Fig. 2).
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We then examined the concentration dependence of the K+-induced current (0.3, 1.0, 3.0, and 10.0 mM extracellular K+) at 50 mV in the mutants that had an outward Na,K-pump current as shown in Fig. 4D. The k1/2 values were 1.18 ± 0.10 mM for the wild-type Na,K-ATPase (n = 8), 0.75 ± 0.06 mM for the K800A mutant (n = 12), and 1.52 ± 0.23 mM for the K800E mutant (n = 8).
We also examined the electrogenic transport activity of the H,K-ATPase mutants in the absence of extracellular Na+ (Fig. 4B), a condition under which the affinity for external K+ is higher (19). Fig. 4E shows that ouabain-sensitive outward currents were recorded in the oocytes expressing the wild-type Na,K-ATPase and those expressing the K800A, K800S, and K800E mutants, whereas there were no such currents in the oocytes injected with the -subunit alone (control group). The K+-induced currents were of similar magnitude as the ouabain-sensitive currents in all these groups except for the K800S mutant, for which a small but significant inward current was induced by 5 mM K+, whereas ouabain addition (in the presence of5mM K+) caused a further inward current shift, demonstrating the existence of an ouabain-sensitive outward current.
The concentration dependence of the K+-induced current (0.02, 0.1, 0.5, 2, and 5 mM extracellular K+) in the absence of extracellular Na+ and at a holding potential of 50 mV is shown in Fig. 4F. These results are analogous to those observed in the presence of extracellular Na+ for the wild-type Na,K-ATPase and the K800A and K800E mutants. The K800A mutant had a slightly lower K+-induced current amplitude, whereas the K800E mutant had a much reduced current amplitude. The apparent affinity for K+ was 0.24 ± 0.12 mM (n = 6) for the Na,K-ATPase, a value similar to that reported earlier under similar conditions (19); 0.23 ± 0.02 mM (n = 11) for the K800A mutant; and 0.73 ± 0.14 mM (n = 6) for the K800E mutant. The K800S mutant showed a more complex K+ concentration/current relationship with a positive (outward) current in the low concentration range, reaching a maximal value of 12.7 ± 0.6 nA (n = 15) recorded at 0.5 mM K+, which reverted to a negative (inward) current at higher concentrations. Fig. 4F also shows the concentration dependence of the inward current induced by K+ in the wild-type H,K-ATPase, a current that is related rather to the intracellular alkalization than to an electrogenic transport activity of this H,K-ATPase as shown earlier (8). The maximal K+-induced current was 25.9 ± 1.8 nA (n = 5), and the apparent affinity for external K+ was 0.12 ± 0.08 mM.
The voltage dependence of the activity of the wild-type Na,K- and H,K-ATPases and of the electrogenic mutants of the H,K-ATPase was also studied in Na+-free extracellular solutions. The currents induced by 5 mM K+ in the wild-type Na,K- and H,K-ATPases and in the K800A and K800E mutants of the H,K-ATPase are shown as a function of the membrane potential in Fig. 5A. For the K800S mutant of the H,K-ATPase, we chose the current induced by 0.5 mM K+, which was the concentration giving the highest outward current. The wild-type Na,K-ATPase current/voltage curve shows little voltage dependence in the 0 to 50 mV membrane potential range and a negative slope in the high negative voltage range, similar to what has been described previously for Na,K-pumps expressed in Xenopus oocytes (6, 19). Potassium induced a voltage-dependent inward current in the wild-type H,K-ATPase, similar to what we have described previously (8). The K800A and K800E mutants were only weakly voltage-dependent, whereas the K800S mutant displayed stronger voltage dependence along the whole membrane potential range (a 4-fold reduction of the ouabain-sensitive current between 10 and 110 mV compared with a <30% decrease for the K800A and K800E mutants over the same voltage range).
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The negative slope of the Na,K-ATPase K+-activated current/voltage curve has been attributed to inhibition by K+ of proton leak conductance in Na+- and K+-free extracellular solutions (24). We examined whether the H,K-ATPase and its electrogenic mutant would present a similar "proton leak." Fig. 5B shows the current sensitive to 2 mM ouabain in a K+- and Na+-free solution at pH 6.0. As shown previously (6, 24), ouabain inhibited a large inward current in the Na,K-ATPase, but there were no such ouabain-sensitive currents in the H,K-ATPase or in any of the mutants.
Electrogenic Transport by the Ser782 Mutants of the Na,K-ATPaseThe stimulation of electrogenic transport by extracellular K+ was also examined in the two Na,K-ATPase mutants (S782A and S782R) that had sizable K+ transport (86Rb uptake) activity. As these mutants had a very low apparent affinity for K+ (see above and Fig. 2), we measured the current inhibited by 2 mM ouabain in 40 mM K+ solution. Under these conditions, as shown in Fig. 6, ouabain inhibited an outward current over the whole potential range in the wild-type Na,K-ATPase and in the S782A mutant, but not in the S782R mutant. The ouabain-sensitive current of the wild-type Na,K-ATPase was voltage-dependent, whereas there was no clear voltage dependence for the S782A mutant over the explored potential range. As both of these mutants had significant 86Rb uptake activity under similar conditions (Fig. 3C), these results indicate that the S782A mutant is electrogenic, but that the S782R mutant has electroneutral cation transport activity.
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DISCUSSION |
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The results of our amino acid substitution generally support our starting hypothesis, viz. that the presence of the positively charged amino acid in the middle of the fifth transmembrane segment is a determinant for the stoichiometry of the cation exchange by the Na,K- and H,K-ATPases. When Ser782 of the Na,K-ATPase was replaced with a positively charged arginine, the mutant was able to transport 86Rb, but showed no electrogenic transport activity, whereas mutation to a neutral alanine induced a reduction of the apparent affinity for K+, but preserved an electrogenic mode of transport. Conversely, mutation of the positively charged Lys800 of the H,K-ATPase to alanine yielded electrogenic transport activity roughly similar in amplitude and apparent affinity for extracellular K+ to that of the Na,K-pump, whereas replacing Lys800 with another positively charged residue, arginine, produced non-electrogenic transport activity. Surprisingly, replacing Lys800 of the H,K-ATPase with a negatively charged glutamic acid yielded significant electrogenic transport activity, which conserved an apparent affinity for extracellular K+ not too different from that of the wild-type Na,K-pump, even though the transport activity could hardly be detected by the less sensitive 86Rb transport assay. Our results do not allow us to draw conclusions about the precise stoichiometry of the cation exchange. It can only be stated that the non-electrogenic H,K-ATPase and mutants must exchange a symmetrical number of cations, whereas the Na,K-ATPase and the mutant that carries an outward current must export out of the cell a larger number of cations than they import into the cell.
The only result discordant with our hypothesis was obtained with the H,K-ATPase mutant in which Lys800 was replaced with serine (K800S), the amino acid found at the homologous position in the Na,K-ATPase. This mutant had robust 86Rb transport activity, but showed no K+-induced or ouabain-sensitive currents at high extracellular Na+ concentrations. However, when studied in the absence of extracellular Na+, this mutant showed a significant ouabain-sensitive outward current at 5 mM K+, whereas the concentration/current relationship shows a biphasic behavior: a small outward current was observed at low K+ concentrations, but it reversed to an inward current at higher K+ concentrations. The ouabain-sensitive outward current indicates that the oocytes injected with the K800S mutant cRNA express an electrogenic pump. The presence of a K+-induced inward current significantly larger than that recorded in oocytes expressing the -subunit alone shows that these oocytes have also another K+-dependent electrogenic pathway linked to expression of the K800S mutant. However, the inward current was not ouabain-sensitive; and thus, it is not due to a normal H,K- or Na,K-pump function. We can hypothesize that this K+-induced inward current is related to a misfolded form of the mutant protein or to any alteration of the oocyte membrane resulting from expression of this mutant, but our data do not allow us to determine the precise cause of this K+-induced inward current. Thus, the K800S mutant seems to have a complex behavior with a stoichiometry that varies according to the presence of extracellular Na+.
The Na,K-ATPase is responsible for an ouabain-sensitive inward current when exposed to Na+- and K+-free extracellular solutions, and this current is increased at acidic pH (6, 24, 33). With Xenopus laevis Na,K-pumps, this current has been shown to be carried, at least in part, by protons (24). This inward flow of protons has no physiological significance because the Na,K-pump is never exposed to extracellular solutions with very low concentrations of Na+ and K+ under physiological conditions. In contrast, the H,K-ATPases are expressed in the apical membrane of epithelial cells and can be exposed to low concentrations of Na+ and K+. We have shown here that neither the non-gastric H,K-ATPase nor any of its electrogenic mutants carry a pH-dependent ouabain-sensitive inward current under these circumstances. Thus, this pH-dependent ouabain-sensitive inward current is not related to the 3:2 stoichiometry of the Na,K-pump.
Taken together, our results point to the crucial role of the highly conserved Ser782 in the Na,K-ATPase and its homologous residue (Lys800) in the H,K-ATPases. Because of the large difference in Na,K-pump transport activity at 5 and 40 mM K+ observed in mutant S782A, our results first confirm earlier studies showing that this residue has a large influence on the apparent K+ affinity of the Na,K-pump (21, 22, 23); more precisely, the S775A mutation of the sheep Na,K-ATPase yields a very low affinity for K+ without changes in the voltage dependence of this affinity (21). This residue is closely associated with the cation pathway through the Na,K-pump as recently shown by its accessibility to water-soluble sulfhydryl reagents when the Na,K-ATPase has been modified to a channel by palytoxin (34).
The high resolution structure of the group IIa P-ATPase SERCA (1) includes 2 calcium ions located close to the middle of the transmembrane part of the protein, between the fourth, fifth, and sixth transmembrane segments, identifying two calcium-binding sites (sites I and II) according to Toyoshima et al. (1). As the Na,K-pump is known to occlude 3 Na+ ions, the "core" of the transmembrane domain of the Na,K-pump must provide three binding sites. It is presently not known where these binding sites are located in the structure of the Na,K-ATPase, but it is reasonable to postulate that two of these three sites are homologous to the calcium sites I and II of SERCA. We propose the hypothesis that Lys800 in the H,K-ATPases provides a fixed positive charge that is the equivalent of the third Na+ ion transported by the Na,K-ATPase. The location of Ser782 and Lys800 in the three-dimensional structures of the Na,K- and H,K-ATPases, respectively, can be estimated by homology from the known high resolution structure of the group IIa P-ATPase SERCA (1, 35). The rather high similarity of the sequence of the fifth transmembrane segment and, in particular, the absolute conservation of Tyr778, Asn783, and Glu786 in this segment allow an unambiguous alignment of the SERCA and Na,K- and H,K-ATPase sequences (Fig. 1). The residue homologous to Na,K-ATPase Ser782 and H,K-ATPase Lys800 is Ser767 in SERCA (rabbit SERCA1, GenBankTM/EBI accession number P04191 [GenBank] and Protein Data Bank code 1EUL [PDB] ). This residue has been implicated in calcium binding by mutagenesis experiments (32) and is located approximately at the same level as the 2 calcium ions and next to the crucial Asn768 that participate directly in the structure of calcium site I and indirectly in the structure of site II (1). The position of Ser782 is illustrated in Fig. 7. According to this hypothesis, the Na,K-ATPase would load 3 Na+ ions from the intracellular solution into three distinct binding sites; two of these sites would have positions analogous to those of calcium sites I and II in SERCA, and the third one would be close to Ser782. In contrast, the H,K-ATPases would load only 2 cations from the intracellular side, the third cation-binding site being occupied by the charge provided by the butyl ammonium side chain of Lys800 acting as a "tethered cation," as has been proposed for the lysine residue of the DEKA motif of the pore of the voltage-gated Na+ channel (36). Removing the Lys800 side chain in the H,K-ATPase would allow it to work similarly to the Na,K-ATPase. Using homology modeling with SERCA, Ogawa and Toyoshima (37) have recently proposed a position for the third Na+ site at a location between TM5, TM6, TM8, and TM9, at about the same level in the membrane as the two other binding sites that were assumed to be homologous to sites I and II of the Ca-ATPase in the E1 (Ca2+-bound) conformation. From TM5, the side chain of Tyr778 (homologous to Tyr796 in the Bufo non-gastric H,K-ATPase; see Fig. 1) participates in the composition of this binding site. The positions of Ser782 and Lys800 studied in the present work are just slightly more than one helix turn deeper in the membrane, and the long side chain of Lys800 could reach the location predicted by Ogawa and Toyoshima (37) for the third Na+ site and place its charged terminal ammonium there. Thus, our present observations and the tethered cation hypothesis are at least compatible with the present knowledge of the structure of the transmembrane part of the Na,K-ATPase.
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FOOTNOTES |
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To whom correspondence should be addressed: Inst. de Pharmacologie et de Toxicologie, Université de Lausanne, Bugnon 27, CH-1005 Lausanne, Switzerland. Tel.: 41-21-692-5362; Fax: 41-21-692-5355; E-mail: Jean-Daniel.Horisberger{at}ipharm.unil.ch.
1 The abbreviations used are: SERCA, sarcoplasmic/endoplasmic reticulum Ca-ATPase; bl H,K-ATPase, B. marinus bladder H,K-ATPase
-subunit;
1 Na,K-ATPase, B. marinus Na,K-ATPase
1-subunit;
bl, B. marinus bladder
-subunit; TM, transmembrane domain.
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ACKNOWLEDGMENTS |
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REFERENCES |
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