Bufo marinus bladder H-K-ATPase carries out electroneutral ion transport

Muriel Burnay, Gilles Crambert, Solange Kharoubi-Hess, Käthi Geering, and Jean-Daniel Horisberger

Institut de Pharmacologie et de Toxicologie, CH-1005 Lausanne, Switzerland


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Bufo marinus bladder H-K-ATPase belongs to the Na-K-ATPase and H-K-ATPase subfamily of oligomeric P-type ATPases and is closely related to rat and human nongastric H-K-ATPases. It has been demonstrated that this ATPase transports K+ into the cell in exchange for protons and sodium ions, but the stoichiometry of this cation exchange is not yet known. We studied the electrogenic properties of B. marinus bladder H-K-ATPase expressed in Xenopus laevis oocytes. In a HEPES-buffered solution, K+ activation of the H-K-ATPase induced a slow-onset inward current that reached an amplitude of ~20 nA after 1-2 min. When measurements were performed in a solution containing 25 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> at a PCO2 of 40 Torr, the negative current activated by K+ was reduced. In noninjected oocytes, intracellular alkalization activated an inward current similar to that due to B. marinus H-K-ATPase. We conclude that the transport activity of the nongastric B. marinus H-K-ATPase is not intrinsically electrogenic but that the inward current observed in oocytes expressing this ion pump is secondary to intracellular alkalization induced by proton transport.

hydrogen-potassium-adenosinetriphosphatase; electrogenicity; stoichiometry; intracellular pH


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

AS DEFINED BY Axelsen and Palmgren (3), the subgroup IIC of the P-type ion-motive ATPases includes the various isoforms of Na-K-ATPase, gastric H-K- ATPase, and the so-called "nongastric" H-K-ATPases (15). Nongastric H-K-ATPases have been cloned from several vertebrate species, including humans (23), rats (10), rabbits (7), guinea pigs (Swissprot Q64392), and Bufo marinus (17). In mammals, these transport proteins have been shown to be involved in K+ balance (22) and are probably located in the apical membrane of colonic and distal renal epithelia (27). Initially, these ATPases were described as H-K-ATPases on the basis of the demonstration of K+ (Rb uptake experiments) and H+ transport (intracellular alkalization and extracellular acidification) (17), but recent experimental evidence indicates that nongastric H-K-ATPases also transport Na+ in exchange for K+ (8, 9, 12).

Although Na-K-ATPase has a well-defined transport stoichiometry of 3Na+/2K+ (2) and gastric H-K-ATPase probably performs a 2H+/2K+ exchange (26), the exact stoichiometry of the cation transport of nongastric H-K-ATPases has never been defined. As a first approach toward the determination of the ion transport stoichiometry of nongastric H-K-ATPases, we have analyzed the electrogenicity of the transport activity of B. marinus bladder H-K-ATPase expressed in Xenopus laevis oocytes. Our results indicate that nongastric H-K-ATPases are not electrogenic and thus exchange a balanced number of charges during each transport cycle.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

cRNA synthesis and expression in X. laevis oocytes. The 3.7 full-length cDNA of the B. marinus H-K-ATPase alpha -subunit (alpha bl) (17) was subcloned into the pSD5 vector containing a 130-bp poly T tail. cRNA was obtained by in vitro transcription (21). Removal of 110 bp of the 5'-untranslated region containing a GC-rich region significantly improved expression of alpha bl-subunits in X. laevis oocytes.

X. laevis oocytes were obtained and prepared as described (11). Oocytes were injected with either 8 ng alpha bl cRNA or 8 ng of the B. marinus Na-K-ATPase alpha 1-subunit (16) cRNA in combination with 1 ng B. marinus bladder beta -subunit (beta bl) (18) cRNA. Other oocytes were injected with the cRNA of the alpha - and beta -subunits of rabbit gastric H-K-ATPase, 12 and 1.5 ng respectively, or with 1.5 mg of cRNA of the beta -subunit alone. The oocytes were then incubated at 19°C in modified Ham's solution (11) until the electrophysiological or flux measurements.

Electrophysiological measurements. Three 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 endogenous X. laevis oocyte Na-K-ATPase (29). The two-electrode, voltage-clamp technique, using a TEV-200 voltage clamp (Dagan, Minneapolis, MN), was applied to analyze putative H-K-ATPase pump currents. Current signals were filtered at 20 Hz and recorded on a Gould chart recorder (model 220, Gould, Cleveland, OH). The intracellular potential was held at -50 mV. Current-voltage (I-V) curves were obtained by applying a series of 500-ms voltage steps ranging from -150 to +30 mV. The H-K or Na-K pumps were activated by addition of 5 mM K+ to a previously K+-free solution or inhibited by addition of 2 mM ouabain, in the presence of 5 mM K+. In a first set of measurements, the bath solution contained (in mM) 92.4 Na+, 0.82 Mg2+, 5 Ba2+, 0.41 Ca2+, 10 tetraethylammonium, 22.46 Cl-, 2.4 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, 80 gluconate, and 10 HEPES (pH 7.4). For the experiments with CO2 and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, the solution contained less gluconate (48 mM) but 25 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, in addition; this solution was bubbled with 5% CO2 and had a pH of 7.4. All experiments were performed at room temperature.

Intracellular pH measurements. Double-barreled pH-sensitive microelectrodes, using the ion exchanger H-ionophore II cocktail A (Fluka), were prepared as described (14). These electrodes had a resistance of 2-10 GOmega . They were calibrated in HEPES-buffered solutions (pH 6.5 and 7.5) immediately before and after each intracellular pH (pHi) measurement. The pH electrodes were used only if they showed a pH response >52 mV/pH unit. pHi was calculated from the voltage read with the pH barrel minus the membrane voltage read from the reference barrel filled with 3 M KCl.

86Rb uptake measurements. 86Rb uptake was performed as previously described (4). In brief, oocytes were injected with either 8 ng alpha bl cRNA or 8 ng B. marinus Na-K-ATPase alpha 1-subunit cRNA in combination with 1 ng beta bl cRNA. After loading of oocytes with Na+ (see above) and their recovery in a solution containing (in mM) 90 NaCl, 2 CaCl2, 5 BaCl2, and 10 MOPS, pH 7.4, they were transferred to a solution containing (in mM) 5 KCl, 90 NaCl, 1 CaCl2, 5 BaCl2, 1 MgCl2, and 10 HEPES, pH 7.4, as well as 0.2 µM ouabain to inhibit the endogenous oocyte Na-K-ATPase. For measurements of 86Rb uptake by alpha bl-beta bl pumps, all solutions contained 10 µM bumetanide. After addition of 5 µCi/ml 86Rb (Amersham), oocytes were incubated for 12 min at room temperature before being washed in a solution containing (in mM) 90 NaCl, 1.0 CaCl2, 1.0 MgCl2, and 10 HEPES, pH 7.4. Individual oocytes were dissolved in 0.5% SDS and counted.

Drugs. DIDS and ouabain were obtained from Sigma.

Statistical analysis. Data are presented as means ± SE. Statistical analysis of the data was performed by a paired Student's t-test when pairs of measurements obtained in the same oocyte were compared or by an unpaired Student's t-test when different groups of oocytes were compared.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Electrogenic activity of the B. marinus Na-K- and H-K-ATPase. The type of beta -subunit that is associated in situ with alpha bl is not known. We coexpressed alpha bl in X. laevis oocytes together with beta bl, which had been cloned from the same organ and was used in earlier studies to analyze the function of this ATPase (17). Sequence comparison indicates that this beta -subunit is the amphibian equivalent of the mammalian beta 2-isoform (13). The B. marinus Na-K-ATPase alpha -subunit was also coexpressed with beta bl to be able to attribute the observed functional differences between H-K- and Na-K- ATPases specifically to the alpha -subunit.

We first evaluated the functional expression of our ATPases using the 86Rb uptake assay. Figure 1 shows that B. marinus bladder H-K-ATPase was functionally expressed at a similar level to that for Na-K-ATPase, with 86Rb uptake values of ~40-50 pmol · min-1 · oocyte-1 above the background uptake observed in control oocytes injected with the beta -subunit alone and expressing only the endogenous X. laevis Na-K-ATPase alpha -subunit.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1.   86Rb uptake by the H-K- and Na-K-ATPases. The uptake of 86Rb was evaluated in Na+-loaded oocytes injected with the Bufo marinus bladder beta -subunit cRNA alone (left, n = 24), Na-K-ATPase alpha - and beta -subunit cRNAs, (middle, n = 24), or H-K-ATPase alpha - and beta -subunit cRNAs (right, n = 18).

To determine the stoichiometry of B. marinus H-K-ATPase, we studied the electrogenic activity of this ion pump compared with that of B. marinus Na-K-ATPase after expression in X. laevis oocytes. As expected, when Na-K-ATPase was stimulated by addition of 5 mM K+, an outward (positive) current was observed (Fig. 2A). In oocytes expressing the B. laevis H-K-ATPase, K+ induced the slow appearance of an inward (negative) current of small amplitude, which was sensitive to ouabain. The I-V curves of the K+-induced current and the ouabain-sensitive current are shown in Fig. 2B. B. marinus bladder H-K-ATPase (17), like B. marinus Na-K-ATPase (16), is moderately sensitive to ouabain. The ouabain-sensitive Na-K pump current was similar to the K+-activated Na-K pump current. On the other hand, in oocytes expressing H-K-ATPases, the amplitude of the ouabain-sensitive current was significantly larger than the K+-induced current. At present, the reason for this difference is not known. It is possible that low levels of extracellular K+ persist in the unstirred layers close to the membrane due to a small K+ leak from the oocyte that maintains some activity of the H-K pump. No current resulting from endogenous oocyte Na-K-ATPase was observed, because this pump had been inhibited by exposure to 0.2 µM ouabain. In another set of experiments, the effect of 5 mM K+ in the absence and in the presence of 2 mM ouabain was compared. In oocytes expressing H-K-ATPase and at -50 mV, 5 mM K+ induced an inward current of 14.5 ± 3.2 nA in the control condition, which was reduced to 5.5 ± 1.6 nA in the presence of 2 mM ouabain (n = 6, P < 0.005, paired Student's t-test).


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2.   K+-activated and ouabain-sensitive currents. A: current traces recorded from an oocyte expressing the B. marinus Na-K-ATPase (top) or bladder H-K-ATPase (bottom). After a stable current reading (at -50 mV) was reached, 5 mM K+ and 30-60 s later, 2 mM ouabain was added to the bathing solution. Activation of Na-K-ATPase produced a large outward current whereas activation of H-K-ATPase was associated with the slow appearance of a small inward current. B: current-voltage (I-V) relationship of the K-induced (filled symbols) and ouabain-sensitive (open symbols) currents in oocytes expressing Na-K-ATPase (triangles) or H-K-ATPase (circles). Values are means ± SE of 9 measurements. Vm, membrane voltage.

As a control experiment for a nonelectrogenic ion pump, we also measured the 86Rb uptake and the K+-induced current in oocytes injected with cRNA of the alpha - and beta -subunits of the rabbit gastric H-K-ATPase and in oocytes injected with gastric H-K-ATPase beta -subunit cRNA alone. As described for the other groups, endogenous oocyte Na-K-ATPase was inhibited by exposure to ouabain, but these oocytes were not loaded with Na+. When compared with the baseline uptake observed in oocytes expressing the beta -subunit alone (2.5 ± 0.2 pmol · min-1 · oocyte-1, n = 11) or in noninjected oocytes (2.0 ± 0.2 pmol · min-1 · oocyte-1, n = 12), coexpression of the gastric H-K-ATPase alpha - and beta -subunits led to a significant increase in 86Rb uptake (17.8 ± 1.1 pmol · min-1 · oocyte-1, n = 18, P < 0.001 for both comparisons). In this latter group, addition of 20 µM SCH-28080 significantly reduced 86Rb to 12.2 ± 0.8 pmol · min-1 · oocyte-1 (n = 21, P < 0.001). In the same experiments, the 86Rb uptake in oocytes expressing the Na-K-ATPase or the nongastric H-K-ATPase amounted to 78 ± 3 (n = 11) and 73 ± 5 pmol · min-1 · oocyte-1 (n = 10), values similar to those observed in the experiments depicted in Fig. 1. In the same batches of oocytes, addition of 5 mM K+ produced a similar small inward current in the gastric H-K-ATPase alpha ,beta -subunit group (4.8 ± 1.0 nA, n = 10) and the beta -subunit alone group (4.1 ± 1.0 nA, n = 7). Addition of 20 µM SCH-28080 to the 5 mM K+ solution did not induce any detectable current change. In the Na-K-ATPase group, K+ induced an outward current of 143 ± 22 nA (n = 7), whereas a small inward current of 11 ± 5 nA (n = 7) was detected in the B. marinus bladder H-K-ATPase group. Thus expression of gastric H-K-ATPase led to a small but significant rubidium transport that was not accompanied by any evidence for any evident electrogenic activity.

H-K-ATPase activity in the presence of CO2-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. The current generated by the H-K-ATPase could be due to its electrogenic activity resulting from the transport of a larger inward than outward number of cations. Alternatively, the inward current could be due to another transporter activated by changes in the cellular ionic composition, i.e., by a modification of the intracellular pH resulting from the H-K-ATPase activity, for example. To distinguish between these two possibilities, we used the following reasoning. If the K+-induced inward current resulted from intracellular alkalization, preventing this alkalization should result in a smaller K+-induced inward current. In contrast, if the inward current were due to an electrogenic H+ (and Na+) exchange for K+, intracellular acidification may increase this current by providing more substrate (protons) to this ion pump. To test this hypothesis, we compared the effects of K+ activation of the H-K pump in a HEPES-buffered solution and in a solution containing 25 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> bubbled with 5% CO2 (pH 7.4). CO2 is expected to produce intracellular acidification after diffusion across the plasma membrane and transformation into carbonic acid by carbonic anhydrase. The intracellular buffering power of the oocyte is also expected to be increased by the presence of CO2 and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. We observed that the inward current resulting from K+ activation of H-K-ATPase was reduced in the presence of the CO2-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solution (see example in Fig. 3A). Figure 3B illustrates the reduction of the K+-induced inward current over the whole potential range. The mean values of the K+-induced inward currents at -50 mV were 11.7 ± 1.6 nA in the absence and 3.7 ± 1.3 nA (n = 9; P < 0.001) in the presence of CO2-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. As shown in Fig. 3C, the effect of CO2-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> was fully reversible, and the K+-induced inward current did not diminish with the time of the experiment. Moreover, the switch from the HEPES-buffered solution to the CO2-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solution induced a large decrease in the baseline inward current, with a mean change of 60 ± 8 nA (n = 9). In contrast to the inward current observed in oocytes expressing H-K-ATPase, the electrogenic activity of Na-K-ATPase expressed in oocytes was barely modified by exposure of the oocytes to the CO2-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solution: 123.5 ± 15.9 nA in the absence and 115.5 ± 15.8 nA in the presence of CO2-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (n = 9; P > 0.7). These results support the hypothesis that the inward current is secondary to a change in pHi. This hypothesis requires two premises: 1) the activation of H-K-ATPase induces an intracellular alkalization and 2) intracellular alkalization induces an inward current. The first assumption has been well established (17), and the second assumption was verified by the following experiments that were performed in native, noninjected oocytes to demonstrate the presence of an endogenous alkalinization-induced inward current.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 3.   Effects of the presence of CO2 and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> on the K+-activated current. A: current trace recorded from an oocyte expressing the B. marinus bladder H-K-ATPase. After activation of the K+-induced current by 5 mM K+ in an air-equilibrated HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free solution, the solution was changed to a CO2-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solution and the K+-current was activated again. B: I-V relationship of the current induced by 5 mM K+ in a HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free (filled symbols) solution and in a CO2-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solution (open symbols). C: reversible effect of CO2-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> on the current induced by 5 mM K+ in oocytes expressing the B. marinus bladder H-K-ATPase. The results show 7 individual values of the holding current at -50 mV, recorded first in a CO2-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free solution (control), then in a CO2-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solution, and then again in the CO2-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free solution. The mean values of the K+-activated current are -13.1 ± 1.3, -6.0 ± 0.6, and -16.6 ± 1.3 nA, respectively. The decrease in the K+-induced current when it passed from the CO2-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free to the CO2-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solution and the increase from the CO2-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> to the CO2-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free solution are both statistically significant with P < 0.001 (n = 7, paired Student's t-test).

Effect of intracellular alkalization on the membrane current. Noninjected oocytes were first incubated in a CO2-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solution for 2 h and placed into the same solution in the measurement chamber. In a first set of experiments, we monitored the effect on pHi of changing the bath to a CO2-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free solution. In a second set of experiments, we analyzed the effect of the same maneuver on inward membrane currents.

As shown in the example in Fig. 4A, removal of CO2-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> produced a sizeable alkalization of the oocyte that was reversible on reexposure to the CO2-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solution. The mean pHi values in the CO2-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solution and 3 min after removal of CO2 and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> were 7.02 ± 0.03 and 7.39 ± 0.05, respectively (n = 8; P < 0.001).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 4.   Effects of CO2-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> removal on intracellular pH and membrane currents in noninjected oocytes. A: original intracellular pH trace (difference between the potential recorded in the ion-selective electrode and the membrane potential) recorded in a noninjected oocyte by means of a double barreled ion-sensitive microelectrode. Electrode impalement and retraction are indicated by the upward- and downward-pointing arrows, respectively. The electrode was calibrated in pH 6.5 and 7.5 solutions immediately before and after the impalement. B: membrane current recorded in a noninjected oocyte during the same maneuver. C: I-V relationship of the currents induced by CO2-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> removal (n = 11), i.e., the difference in current measured before and 3 min after CO2-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> removal.

Figure 4B shows that pHi changes were paralleled by the appearance of an inward current on CO2-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> removal. At a membrane potential of -50 mV, the amplitude of this current amounted to -113.6 ± 6.4 nA (n = 11). The shape of the I-V relationship of this current was similar to that of the K-induced current observed in oocytes expressing H-K-ATPase (compare Figs. 3B and 4C).

Effect of DIDS on the alkalinization-induced current. Many ion transport systems could be affected by intracellular alkalization to produce the inward current observed after CO2-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> removal or after activation of H-K-ATPase in X. laevis oocytes. Even though endogenous activity of Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport is apparently low in X. laevis oocytes (28), we considered the implication of this electrogenic transport system by testing the effect of DIDS, a known inhibitor of the Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter. DIDS, at a concentration of 500 µM, did not inhibit the K-induced inward current in oocytes expressing H-K-ATPase. The mean K-induced currents were -28.1 ± 7.60 nA before and -28.5 ± 6.18 nA after DIDS treatment (n = 10).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The activity of B. marinus bladder H-K-ATPase is associated with a small inward current, as demonstrated by the effect of K+ activation and ouabain inhibition of this cation pump. By themselves, these results indicate that the stoichiometry of the nongastric H-K pumps is different from that of the Na-K pump, for which a large outward current is observed.

The slow time course of the appearance of the inward current after K+ activation, or of its disappearance after ouabain inhibition, suggests that it is not intrinsically due to the activity of H-K-ATPase but rather results from changes in intracellular ionic conditions due to the ion pump function. We tested the hypothesis that the inward current is due to the activation of an electrogenic process by intracellular alkalization produced by the activity of the H-K pump. We could demonstrate that intracellular alkalization produced by CO2-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> removal is indeed accompanied by the appearance of an inward current in noninjected oocytes. This current and the K+-induced current in oocytes expressing H-K-ATPase have a similar I-V relationship. It should be noted that the pHi, measured by means of ion-sensitive microelectrodes, reflects the bulk pHi of the oocyte. Analogous to sizeable effects of submembranous Na+ concentrations in X. laevis oocytes previously observed (1), it is certainly possible that the sudden activation of the highly expressed H-K-ATPases could result in larger local pH changes.

From the existence of the alkalization-induced inward current and the intracellular alkalization resulting from H-K-ATPase activity, we conclude that the inward current associated with the activity is essentially due to this mechanism.

We have not yet determined the nature of the electrogenic process activated by intracellular alkalization. The I-V relationship of the current indicates a reversal potential close to 0 mV (see Figs. 2 and 4). This is not compatible with the activation of either a K+-selective or a Na+-selective channel under our experimental conditions. Our negative results with a Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport inhibitor also argue against the implication of such a transport system, although it cannot definitively be excluded that a DIDS-resistant Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter exists in oocytes.

Similar to what has been described earlier (20), expression of gastric H-K-ATPase resulted in a lower expression of transport function (assayed as 86Rb uptake) than that obtained for either Na-K-ATPase or nongastric H-K-ATPase. However, this level of expression should have allowed us to detect a K+-induced or SCH-28080-sensitive activity if the activity of this ion pump was electrogenic in a way similar to that of Na-K-ATPase. However, the low level of expression may have prevented us from detecting an alkalization-induced inward current similar to what we observed with nongastric H-K-ATPase. Thus our results confirm the hypothesis of an exchange of a symmetrical number of H+ and K+ ions by gastric H-K-ATPase (26).

Our results are thus compatible with the hypothesis that the activity of B. marinus bladder H-K-ATPase is not intrinsically electrogenic. In this regard, the bladder H-K pump is similar to the gastric H-K pump. What might be the actual ion stoichiometry of this transport system? The available evidence for all the highly related members of the H-K- and Na-K-ATPase family indicates that two K+ ions are transported into the cell for each hydrolyzed ATP, and we make the assumption that this is also the case for the nongastric H-K-ATPases. Because Na+ as well as protons seem to be transported by these pumps (9, 12), the simplest hypothesis is that one H+ and one Na+ ion are exchanged for two K+ ions, but this hypothesis has yet to be supported by experimental data. However, because both Na-K-ATPase and the gastric H-K-ATPase show some flexibility with regard to the stoichiometry of transported ions (5, 6, 24, 25), it is certainly possible that the ratio of Na+ ions to H+ ions is not fixed but varies, depending on the intracellular concentrations of both of these ions.


    ACKNOWLEDGEMENTS

This work was supported by Fonds National Suisse de la Recherche Scientifique Grant 31-45867.95.


    FOOTNOTES

Address for reprint requests and other correspondence: J.-D. Horisberger, Institut de Pharmacologie et de Toxicologie, Bugnon 27, CH-1005 Lausanne, Switzerland (E-mail: Jean-Daniel.Horisberger{at}ipharm.unil.ch).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 13 November 2000; accepted in final form 2 July 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abriel, H, and Horisberger JD. Feedback inhibition of amiloride-sensitive epithelial sodium channel in Xenopus leavis oocytes. J Physiol(Lond) 516: 31-43, 1999[Abstract/Free Full Text].

2.   Apell, HJ. Electrogenic properties of the Na,K pump. J Membr Biol 110: 103-114, 1989[ISI][Medline].

3.   Axelsen, KB, and Palmgren MG. Evolution of substrate specificities in the P-type ATPase superfamily. J Mol Evol 46: 84-101, 1998[ISI][Medline].

4.   Beggah, AT, Jaunin P, and Geering K. Role of glycosylation and disulfide bond formation in the beta  subunit in the folding and functional expression of Na,K-ATPase. J Biol Chem 272: 10318-10326, 1997[Abstract/Free Full Text].

5.   Blostein, R. Proton-activated rubidium transport catalyzed by the sodium pump. J Biol Chem 260: 829-833, 1985[Abstract/Free Full Text].

6.   Blostein, R, and Harvey WJ. Na+, K+-pump stoichiometry and coupling in inside-out vesicles from red blood cell membranes. Methods Enzymol 173: 377-380, 1989[ISI][Medline].

7.   Campbell, WG, Weiner ID, Wingo CS, and Cain BD. H-K-ATPase in the rcct-28a rabbit cortical collecting duct cell line. Am J Physiol Renal Physiol 276: F237-F245, 1999[Abstract/Free Full Text].

8.   Codina, J, Pressley TA, and Dubose TD. The colonic H+,K+-ATPase functions as a Na+-dependent K+(NH<UP><SUB>4</SUB><SUP>+</SUP></UP>)-ATPase in apical membranes from rat distal colon. J Biol Chem 274: 19693-19698, 1999[Abstract/Free Full Text].

9.   Cougnon, M, Bouyer P, Planelles G, and Jaisser F. Does the colonic H,K-ATPase also act as an Na,K-ATPase? Proc Natl Acad Sci USA 95: 6516-6520, 1998[Abstract/Free Full Text].

10.   Crowson, MS, and Shull GE. Isolation and characterization of a cDNA encoding the putative distal colon H+,K+-ATPase. Similarity of deduced amino acid sequence to gastric H+,K+-ATPase and Na +,K+- ATPase and mRNA expression in distal colon, kidney, and uterus. J Biol Chem 267: 13740-13748, 1992[Abstract/Free Full Text].

11.   Geering, K, Theulaz I, Verrey F, Häuptle MT, and Rossier BC. A role for the beta -subunit in the expression of functional Na+-K+-ATPase in Xenopus oocytes. Am J Physiol Cell Physiol 257: C851-C858, 1989[Abstract/Free Full Text].

12.   Grishin, AV, and Caplan MJ. Atp1al1, a member of the non-gastric H,K-ATPase family, functions as a sodium pump. J Biol Chem 273: 27772-27778, 1998[Abstract/Free Full Text].

13.   Horisberger, JD, and Doucet A. Renal ion-translocating ATPases: the P-type family. In: The Kidney, edited by Seldin DW, and Giebisch G.. Philadelphia, PA: Lippincott Williams & Wilkins, 2000, p. 139-170.

14.   Horisberger, JD, and Giebisch G. Intracellular Na+ and K+ activities and membrane conductances in the collecting tubule of Amphiuma. J Gen Physiol 92: 643-665, 1988[Abstract].

15.   Jaisser, F, and Beggah AT. The nongastric H +-K+-ATPases: molecular and functional properties. Am J Physiol Renal Physiol 276: F812-F824, 1999[Abstract/Free Full Text].

16.   Jaisser, F, Canessa CM, Horisberger JD, and Rossier BC. Primary sequence and functional expression of a novel ouabain-resistant Na,K-ATPase. J Biol Chem 267: 16895-16903, 1992[Abstract/Free Full Text].

17.   Jaisser, F, Horisberger JD, Geering K, and Rossier BC. Mechanisms of urinary K+ and H+ excretion: primary structure and functional expression of a novel H,K-ATPase. J Cell Biol 123: 1421-1429, 1993[Abstract].

18.   Jaisser, F, Horisberger JD, and Rossier BC. Primary sequence and functional expression of a novel beta  subunit of the P-ATPase gene family. Pflügers Arch 425: 446-452, 1993[ISI][Medline].

19.   Jaisser, F, Jaunin P, Geering K, Rossier BC, and Horisberger JD. Modulation of the Na,K-pump function by the beta -subunit isoforms. J Gen Physiol 103: 605-623, 1994[Abstract].

20.   Mathews, PM, Claeys D, Jaisser F, Geering K, Horisberger JD, Kraehenbühl JP, and Rossier BC. Primary structure and functional expression of the mouse and frog alpha -subunit of the gastric H+-K+-ATPase. Am J Physiol Cell Physiol 268: C1207-C1214, 1995[Abstract/Free Full Text].

21.   Melton, DA, Krieg PA, Rebagliati MR, Maniatis T, Zinn K, and Green MR. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res 12: 7035-7056, 1984[Abstract].

22.   Meneton, P, Schultheis PJ, Greeb J, Nieman ML, Liu LH, Clarke LL, Duffy JJ, Doetschman T, Lorenz JM, and Shull GE. Increased sensitivity to K+ deprivation in colonic H,K-ATPase-deficient mice. J Clin Invest 101: 536-542, 1998[Abstract/Free Full Text].

23.   Modyanov, NN, Mathews PM, Grishin AV, Beguin P, Beggah AT, Rossier BC, Horisberger JD, and Geering K. Human ATP1al1 gene encodes a ouabain-sensitive H-K-ATPase. Am J Physiol Cell Physiol 269: C992-C997, 1995[Abstract/Free Full Text].

24.   Polvani, C, and Blostein R. Protons as substitutes for sodium and potassium in the sodium pump reaction. J Biol Chem 263: 16757-16763, 1988[Abstract/Free Full Text].

25.   Polvani, C, and Blostein R. Effects of cytoplasmic sodium concentration on the electrogenicity of the sodium pump. J Biol Chem 264: 15182-15185, 1989[Abstract/Free Full Text].

26.   Rabon, EC, and Reuben MA. The mechanism and structure of the gastric H,K-ATPase. Annu Rev Physiol 52: 321-344, 1990[ISI][Medline].

27.   Rajendran, VM, Sangan P, Geibel J, and Binder HJ. Ouabain-sensitive H,K-ATPase functions as Na,H-ATPase in apical membranes of rat distal colon. J Biol Chem 275: 13035-13040, 2000[Abstract/Free Full Text].

28.   Romero, MF, Hediger MA, Boulpaep EL, and Boron WF. Expression cloning and characterization of a renal electrogenic Na+/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter. Nature 387: 409-413, 1997[ISI][Medline].

29.   Wang, X, Jaisser F, and Horisberger JD. Role in cation translocation of the N-terminus of the alpha -subunit of the Na+,K+-pump of Bufo. J Physiol (Lond) 491: 579-594, 1996[Abstract].


Am J Physiol Renal Fluid Electrolyte Physiol 281(5):F869-F874
0363-6127/01 $5.00 Copyright © 2001 the American Physiological Society