Institut de Pharmacologie et de Toxicologie, CH-1005 Lausanne, Switzerland
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ABSTRACT |
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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
hydrogen-potassium-adenosinetriphosphatase; electrogenicity; stoichiometry; intracellular pH
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INTRODUCTION |
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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.
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METHODS |
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cRNA synthesis and expression in X. laevis oocytes.
The 3.7 full-length cDNA of the B. marinus H-K-ATPase
-subunit (
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
bl-subunits in X. laevis
oocytes.
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
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 G. 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
bl cRNA or 8 ng B. marinus Na-K-ATPase
1-subunit cRNA in combination with 1 ng
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
bl-
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.
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RESULTS |
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Electrogenic activity of the B. marinus
Na-K- and H-K-ATPase.
The type of -subunit that is associated in situ with
bl is not known. We coexpressed
bl in
X. laevis oocytes together with
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
-subunit is the amphibian equivalent
of the mammalian
2-isoform (13). The
B. marinus Na-K-ATPase
-subunit was also coexpressed with
bl to be able to attribute the observed functional
differences between H-K- and Na-K- ATPases specifically to the
-subunit.
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H-K-ATPase activity in the presence of
CO2-HCO50 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
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Effect of intracellular alkalization on the membrane current.
Noninjected oocytes were first incubated in a
CO2-HCO
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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-HCO28.1 ± 7.60 nA before and
28.5 ± 6.18 nA after DIDS
treatment (n = 10).
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DISCUSSION |
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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
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
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.
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ACKNOWLEDGEMENTS |
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This work was supported by Fonds National Suisse de la Recherche Scientifique Grant 31-45867.95.
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FOOTNOTES |
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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.
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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
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 subunit in the folding and functional expression of Na,K-ATPase.
J Biol Chem
272:
10318-10326,
1997
5.
Blostein, R.
Proton-activated rubidium transport catalyzed by the sodium pump.
J Biol Chem
260:
829-833,
1985
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
8.
Codina, J,
Pressley TA,
and
Dubose TD.
The colonic H+,K+-ATPase functions as a Na+-dependent K+(NH
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
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
11.
Geering, K,
Theulaz I,
Verrey F,
Häuptle MT,
and
Rossier BC.
A role for the -subunit in the expression of functional Na+-K+-ATPase in Xenopus oocytes.
Am J Physiol Cell Physiol
257:
C851-C858,
1989
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
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
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
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 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 -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 -subunit of the gastric H+-K+-ATPase.
Am J Physiol Cell Physiol
268:
C1207-C1214,
1995
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
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
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
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
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
28.
Romero, MF,
Hediger MA,
Boulpaep EL,
and
Boron WF.
Expression cloning and characterization of a renal electrogenic Na+/HCO
29.
Wang, X,
Jaisser F,
and
Horisberger JD.
Role in cation translocation of the N-terminus of the -subunit of the Na+,K+-pump of Bufo.
J Physiol (Lond)
491:
579-594,
1996[Abstract].