1 Institute Of Pharmacology And Toxicology of The University, CH-1005 Lausanne, Switzerland; and 2 Department Of Pharmacology, Medical College Of Ohio, Toledo, Ohio 43614-5804
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ABSTRACT |
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To
investigate whether nongastric H+-K+-ATPases
transport Na+ in exchange for K+ and whether
different -isoforms influence their transport properties, we
compared the functional properties of the catalytic subunit of human
nongastric H+-K+-ATPase, ATP1al1 (AL1), and of
the Na+-K+-ATPase
1-subunit
(
1) expressed in Xenopus oocytes, with
different
-subunits. Our results show that
HK and
1-NK can produce functional AL1/
complexes at the
oocyte cell surface that, in contrast to
1/
1 NK and
1/
HK
complexes, exhibit a similar apparent K+ affinity. Similar
to Na+-K+-ATPase, AL1/
complexes are able to
decrease intracellular Na+ concentrations in
Na+-loaded oocytes, and their K+ transport
depends on intra- and extracellular Na+ concentrations.
Finally, controlled trypsinolysis reveals that
-isoforms influence
the protease sensitivity of AL1 and
1 and that AL1/
complexes, similar to the Na+-K+-ATPase, can
undergo distinct K+-Na+- and ouabain-dependent
conformational changes. These results provide new evidence that the
human nongastric H+-K+-ATPase interacts with
and transports Na+ in exchange for K+ and that
-isoforms have a distinct effect on the overall structural integrity
of AL1 but influence its transport properties less than those of the
Na+-K+-ATPase
-subunit.
X+-K+-ATPases; Na+ transport; Xenopus oocytes; intersubunit interactions
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INTRODUCTION |
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THE CATALYTIC SUBUNIT
of the human nongastric H+-K+-ATPase is termed
ATP1al1 (AL1) (35, 43, 46). This enzyme is a
member of the closely related X+-K+-ATPases of
the P-type ATPase family, which transport K+ in exchange
for Na+ and/or H+. Based on their structural,
functional, and pharmacological properties, animal nongastric
H+-K+-ATPases (for recent review and references
see Ref. 20) occupy an intermediate position between
Na+-K+-ATPase and gastric
H+-K+-ATPase. For instance, their -subunits
are structurally equally distant from
Na+-K+-ATPase and gastric
H+-K+-ATPase
-subunits, and the nongastric
H+-K+- ATPases are moderately sensitive to
the Na+-K+- ATPase inhibitor, ouabain, and
poorly sensitive to the gastric H+-K+-ATPase
inhibitor, SCH-28080 (9, 16, 22, 34). Similar to
Na+-K+-ATPase and gastric
H+-K+-ATPase, nongastric
H+-K+-ATPases are oligomeric P-type ATPases
that need a
-subunit for the structural maturation of the catalytic
-subunit (for review see Ref. 13). However, it is
presently not known which of the four known
X+-K+-ATPase
-subunits
(Na+-K+-ATPase
1-,
2-, and
3-isoforms and gastric
H+-K+-ATPase
-subunit) is associated with
the nongastric H+-K+-ATPase
-subunit in situ.
In mammals, nongastric H+-K+-ATPase -mRNA is
widely distributed, with prominent expression in skin, colon, and
kidney (37). At the protein level, the nongastric
H+-K+-ATPase
-subunit has been identified in
apical membranes of surface cells in distal colon (30) and
of principal cells in the outer medullary collecting duct of rat kidney
(41) or of connecting tubule cells of rabbit kidney
(12). In humans, the AL1 protein was detected in
intercalated and, to a lesser extent, in principal cells of the renal
collecting ducts (28). mRNA and protein abundance is
significantly upregulated by dietary Na+ depletion in the
rat and mouse colon and by dietary K+ depletion in the rat
kidney (23, 31, 41, 45). Although these adaptive,
regulatory mechanisms and findings from mice deficient in nongastric
H+-K+-ATPase
-subunits (33, 45)
suggest that nongastric H+-K+-ATPases play a
role in the maintenance of K+ homeostasis in vivo under
K+- or Na+-deprived conditions, the exact
biological function of these ATPases is still unclear, mainly because
of the still insufficient characterization of their functional
properties and of subunit composition.
The capacity of different, putative nongastric
H+-K+-ATPases to transport K+,
H+, and NH
complexes, respectively. The Na+ transport capacity of
nongastric H+-K+-ATPase is still ill defined.
For instance, nothing is known on the Na+ dependence of the
K+ transport and the actual affinity for intracellular
Na+.
For their functional expression, nongastric
H+-K+-ATPase -subunits depend on the
presence of a
-subunit (1, 2, 16, 22, 34, 39). Both
Na+-K+-ATPase
1-subunit
(6, 29) and
3-subunit (40)
have been proposed as the natural partner subunit of nongastric
H+-K+-ATPase
-subunits in kidney and colon.
In expression systems, several
-subunits, including the
Na+-K+-ATPase
1-subunit
(2, 7, 9), the Na+-K+-ATPase
2-like Bufo bladder
-subunit
(11), a Torpedo
-subunit (2),
as well as the gastric H+-K+-ATPase
-subunit
(1, 7, 16, 21, 27, 34) can support the functional
expression of nongastric H+-K+-ATPases. On the
other hand, in HEK-293 cells, nongastric
H+-K+-ATPases cannot assemble with endogenous,
probably
1-subunits (2, 39), and in
Xenopus oocytes only gastric
H+-K+-ATPase
-subunits and
Na+-K+-ATPase
2 and
2-like Bufo bladder
-subunits can promote
efficient maturation of
-subunits of nongastric
H+-K+-ATPase such as AL1 (15).
Altogether, these apparently contradictory results reflect the
uncertainty about the real, physiologically significant subunit
composition of nongastric H+-K+-ATPase. This
may be of relevance for the interpretation of functional properties
of nongastric H+-K+- ATPases studied in
expression systems, in terms of their physiological importance. Indeed,
-subunits are not only specific chaperones necessary for the
maturation of
-subunits of X+-K+-ATPases,
but they were also shown to modulate the transport properties and, in
particular, the K+ activation of
Na+-K+-ATPase and gastric
H+-K+-ATPase (for review see Ref.
13).
The present study aimed at a further characterization of the functional
properties of the human nongastric
H+-K+-ATPase, in particular of its
K+/Na+ transport capacity, and at a better
understanding of the influence of different -subunits on various
functional parameters. For this purpose, we expressed AL1 or
Na+-K+-ATPase
-subunits together with
Na+-K+-ATPase
1-subunit, gastric
H+-K+-ATPase
-subunit, or Bufo
bladder
-subunit in Xenopus oocytes and compared the
Na+ and K+ dependence of the transport activity
of the various pumps and the ligand-dependent conformational changes of
the
-associated
-subunits, as reflected by controlled
proteolysis. Our results provide further evidence for a
Na+/K+ exchange mode of AL1. Moreover, our
findings indicate that
-isoforms indeed influence the overall
structural integrity of AL1 but, compared with the
Na+-K+-ATPase, have less effects on the cation
transport properties of the human nongastric
H+-K+-ATPase.
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MATERIALS AND METHODS |
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Preparation of expression constructs and cRNAs.
The expression constructs of AL1 in the pSD3 vector and of human
Na+-K+-ATPase 1-subunits
(
1) in the pNKS2 vector have been prepared as described
previously (34). Human
Na+- K+-ATPase
1-subunits
(
1) (26) (kindly provided by K. Kawakami), rabbit gastric H+-K+-ATPase
-subunit
(
HK), and
-subunit (
HK; kindly provided by G. Sachs),
Bufo Na+-K+-ATPase
1-subunits (Bufo
1)
(21), Bufo bladder
-subunits (
bl; kindly
provided by F. Jaisser) (22), and
-,
-, and
-subunits of the rat epithelial Na+ channel (rENaC)
(5) were subcloned in the pSD5 vector. cRNAs were prepared
by in vitro transcription (32).
Protein expression in Xenopus oocytes, controlled proteolysis,
and immunopreciptations.
Oocytes were obtained from Xenopus laevis females
as described before (14). Routinely, 8 ng of -cRNA and
1 ng of
-cRNA were injected, and the oocytes were incubated in the
presence of 0.8 mCi/ml [35S]Easytag express protein
labeling mix (New England Nuclear) and in the absence or presence of 5 µg/ml brefeldin A, which retains proteins in the endoplasmic
reticulum (14). After a 24-h pulse period at 19°C,
microsomal fractions were prepared as described (14) in
the absence of protease inhibitors, and the pelleted membranes were
taken up in a solution containing 30 mM DL-histidine, 5 mM
EDTA, and 18 mM Tris, pH 7.4. Aliquots of 12 µg protein were incubated in the presence of 4 mM MgCl2, 50 mM NaCl, 15 mM
KCl, 1 or 4 mM Tris-ATP (Sigma Chemical), 1.5 mM ouabain, and 7.5 mM Tris-Pi, alone or in combination, as indicated in figure
legends. Osmolarity was adjusted to 59 mM with choline chloride.
Samples were preincubated for 30 min at 25°C before addition of
diphenyl carbamyl chloride-treated trypsin (Sigma), at a trypsin to
protein ratio of 0.05. Samples were incubated for 1 h at 25°C
before addition of a fivefold excess over trypsin (wt/wt) of soybean
trypsin inhibitor (Sigma). After 10 min at 25°C, SDS was added to a
final concentration of 3.7% and the samples were heated at 56°C for
7 min. Immunoprecipitations of
- and
-subunits were performed
with specific antibodies under denaturing conditions as previously
described (14). In some instances, samples were treated
with endoglycosidase H (Endo H; Biolab) (14), to remove
core sugars from
-subunits, and subjected to SDS-PAGE without
immunoprecipitation. Immunoprecipitated and nonimmunoprecipitated
-
and
-subunits were detected by fluorography. Quantification of
immunoprecipitated
-subunits was performed with a laser densitometer
(LKB Ultroscan 2202). Statistical analysis was performed with unpaired
Student's t-test.
Measurement of
[Na+]i.
Oocytes were injected with 0.3 ng each of -,
-, and
-subunit
cRNAs of rENaC and 1 ng of
1- or
HK cRNA with or
without 8 ng of Bufo
1, AL1,
HK, or
1-cRNA and incubated in modified Barth's solution
containing 90 mM NaCl. Determinations of
[Na+]i were performed 3 days after injection
using the two-electrode voltage-clamp technique as described before
(18). The day before the measurements, oocytes expressing
the moderately ouabain-resistant Bufo
1/
,
AL1/
, and the ouabain-resistant
HK/
complexes were incubated
with 1 µM ouabain to inhibit the endogenous oocyte
Na+-K+-ATPase. [Na+]i
was calculated from the reversal potential of the amiloride-sensitive current mediated by the coexpressed rENaC and deduced from
current-voltage curves recorded in the absence or presence of 20 µM
amiloride in a solution containing 5 mM sodium gluconate, 0.5 mM
MgCl2, 2.5 mM BaCl2, 95 mM
N-methyl-D-glucamine (NMDG)-Cl, and 10 mM NMDG-HEPES, pH 7.4.
Determination of the apparent affinity for intracellular
Na+ of AL1/ complexes.
Immediately after injection with X+-K+-ATPase
and rENaC subunit cRNAs, oocytes were incubated in the absence of
K+ in modified Barth's solution containing 0, 5, 20, and
100 mM sodium gluconate. In each solution, the osmolarity was adjusted to 100 mM with choline chloride. After 3 days of incubation,
[Na+]i was determined as described above.
Measurement of 86Rb uptake was carried out as described
before (34) by transferring oocytes into a solution
containing 90 mM NaCl, 2 mM CaCl2, 5 mM BaCl2,
1 mM MgCl2, 10 mM HEPES, pH 7.4, 5 µM amiloride, 10 µM bumetamide, 1 µM ouabain, and 5 µCi/ml 86Rb (Amersham)
for 12 min at room temperature before washing three times in a solution
containing 90 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES, pH 7.4. Individual oocytes were dissolved in 0.5% SDS
and counted.
Measurements of pHi.
pHi measurements were carried out by using a
double-barreled pH-sensitive microelectrode containing ion exchanger
H-ionophore II cocktail A (Fluka) as previously described
(19). These electrodes had a resistance of 2-10 G
and were calibrated in HEPES buffer solutions (pH 6.5 and 7.5)
immediately before and after each 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.
Determination of the apparent affinity for K
+ and of the effect of extracellular
Na+ on the
K+ transport of AL1/ complexes.
86Rb uptake was measured 3 days after injection of oocytes
with AL1 together with
1 or
HK cRNAs. To determine
the K+ activation constant of AL1/
1 and AL1/
HK
complexes, oocytes were loaded with Na+ as described before
(24), and the 86Rb uptake was measured in the
presence of various K+ concentrations (0.2, 0.3, 0.5, 1, 3, and 5 mM) in a solution containing 90 mM NaCl, 2 mM CaCl2,
5 mM BaCl2, 1 mM MgCl2, 10 µM bumetamide, 1 µM ouabain, and 5 µCi/ml 86Rb as described above. To
determine the effect of extracellular Na+ on the
K+ transport of AL1/
1 and AL1/
HK
complexes, oocytes were loaded with Na+, and
86Rb uptake was measured in a solution containing 0.5 mM
KCl and either 90 mM NMDG-Cl or 90 mM NaCl.
Electrophysiological measurements of
Na+-K+
pumps.
Na+-K+ pump currents were measured as the
K+-induced outward current using the two-electrode
voltage-clamp technique as described before (24). Briefly,
current measurements were performed 3 days after injection of oocytes
with 1 cRNAs together with
1 or
HK. To
determine the maximal K+-induced current, injected and
noninjected oocytes were loaded with Na+ in a
K+-free solution 1 day before measurements. The currents
induced by addition of 10 mM K+ were recorded at
50 mV in
a Na+-containing solution (80 mM sodium gluconate, 0.82 mM
MgCl2, 0.41 mM CaCl2, 5 mM BaCl2,
10 mM tetraethylammonium chloride, and 10 mM NMDG-HEPES, pH 7.4). To
determine the K+ activation of
Na+-K+ pumps, the pump current was measured in
the presence of 0.3, 1, 3, and 10 mM K+. The Hill equation,
IK = Imax/{1 + (K1/2
K+/[K])nH}, was fitted to the
current (IK) induced at different K+
concentrations ([K]) to yield least-square estimates of the maximal current (Imax) and of the half-activation
constant for K+ (K1/2
K+). A Hill coefficient (nH) of 1.6 was used as previously described (24). The K+
activation of pump currents mediated by Na+-K+
pumps expressed in oocytes was determined after subtracting the currents due to endogenous oocyte Na+-K+ pumps.
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RESULTS |
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Functional cell surface expression of AL1 or
Na+-K+-ATPase
1 associated with different
-subunits.
By using the Xenopus oocyte as an expression system, we have
previously established (for review see Ref. 13) that all
-subunits can act as chaperones for the structural maturation of
Na+-K+-ATPase
1-subunits
(
1). On the other hand, only the gastric H+-K+-ATPase
-subunit (
HK) and the
Na+-K+-ATPase
2-like
Bufo bladder
-subunit (
bl), but not the
Na+-K+-ATPase
1-subunit
(
1), can efficiently associate with AL1 and form stable
AL1/
complexes (15). In the present study, we have tested the cell surface expression and transport properties of AL1 and
1 associated with different
-subunits. As revealed by electrophysiological measurements, coexpression of
HK with
1 led to a reduced Na+-K+ pump
activity compared with that produced after coexpression with
1 (Fig. 1A,
compare lanes 1 and 2), which, as shown
below, is likely due to the lower apparent K+ affinity of
1/
HK complexes. Similar to results obtained with nongastric Bufo bladder H+-K+-ATPase
(4), the transport activity of AL1/
HK complexes could not be revealed by electrophysiological measurements (data not shown)
due to lack of electrogenicity. On the other hand, 86Rb
uptake measurements showed that, despite its moderate association efficiency,
1 produces a number of
K+-transporting AL1 complexes at the cell surface similar
to
HK or
bl (Fig. 1B, lanes 1,
3, and 4). As previously described (34), 1 mM ouabain partially inhibited the transport
function of AL1/
complexes (lane 2).
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-Isoforms differentially influence the
K+ activation of
Na+-K+
pumps but not of nongastric
H+-K+
pumps.
To reveal a possible influence of
-subunits on the transport
properties of AL1, we compared the K+ activation of pump
activities mediated by
1 or AL1 coexpressed with
different
-subunits in Xenopus oocytes. As shown in Fig. 2 and in previous studies (18,
22),
1 associated with
HK produced
Na+-K+ pumps with a significantly reduced
K+ affinity compared with that of
1
associated with
1 (Fig. 2, lanes 3 and
4). On the other hand, the K1/2 value
for K+ was similar for AL1 associated with
HK or
1 (Fig. 2, lanes 1 and 2),
indicating that the K+ effect of
-isoforms is less
pronounced in nongastric H+-K+-ATPase than in
Na+-K+-ATPase.
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Na+ transport function of AL1 and
1/
complexes.
It has been reported that expression of colonic
H+-K+-ATPase in Xenopus oocytes
(10) or of AL1 in HEK cells (17) lowers [Na+]i, suggesting that nongastric
H+-K+-ATPases may transport Na+ in
exchange for K+. We compared the Na+ transport
activity of AL1/
HK, AL1/
1, and
1/
1 complexes in oocytes in which
[Na+]i was increased by the coexpression of
the amiloride-sensitive epithelial Na+ channel.
[Na+]i amounted to ~80 or 20 mM in oocytes
expressing
-subunits alone and treated (Fig.
3A, lane 1) or not
(Fig. 3B, lane 1) with 1 µM ouabain to inhibit
endogenous oocyte Na+-K+-ATPase. Similar to the
colonic H+-K+-ATPase
-subunit coexpressed
with
HK (17), oocyte-expressed AL1/
HK (Fig.
3A) or AL1/
1 (data not shown) complexes
produced a significant, more than twofold decrease in the intracellular Na+ content (compare lane 3 to lane
1), which was comparable to that produced by Bufo
1/
1 complexes (lane 2) or by
human
1/
1 complexes (Fig. 3B,
compare lane 2 with lane 1). The specificity of
the observed effect is supported by the fact that gastric
HK/
HK complexes did not decrease significantly
[Na+]i compared with control oocytes, despite
an expression similar to that of AL1/
complexes (data not shown).
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Controlled proteolysis to probe the ligand dependence of AL1/,
1/
, and
1-AL1/
complexes.
By using purified Na+-K+-ATPase preparations,
it was previously shown that the proteolytic sensitivity and the
production of specific proteolytic fragments of the
-subunit change
in the presence of different ligands, as a reflection of its
ligand-dependent conformational changes (25). We developed
a controlled trypsinolysis assay to further characterize the substrate
dependence of AL1 and to compare the influence of different
-subunits on the functional properties of
1 and AL1
proteins expressed in Xenopus oocytes. Because the
antibodies used to detect
1, and in particular AL1, did
not efficiently recognize proteolytic
-fragments, the analysis was
mainly based on the extent of the overall proteolytic sensitivity of
the
-subunits in the presence of different ligands. With the exception of a small shift in the molecular mass of
1,
-subunits were not significantly digested in any of the proteolysis
conditions (Fig. 4, A and
B, lanes 10-18). On the other hand, as expected (14), all
-subunits expressed without a
-subunit
were completely digested by trypsin under all conditions (data not
shown).
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K+-dependent conformational changes
of AL1 in the presence of different -subunits.
To analyze whether the well-established ATP-dependent K+
transport of nongastric H+-K+-ATPases is
reflected in ligand-dependent conformational changes of AL1, we
performed controlled proteolysis assays on AL1 associated with
different
-subunits in the presence of K+,
Mg2+, and/or ATP. In general, AL1 associated with
HK was
more trypsin sensitive than AL1 associated with
bl or
1 (Fig. 5,
A-E). This result compares with the higher trypsin
sensitivity of
1/
HK than of
1/
1 complexes (compare Fig. 4C
and Fig. 5, D and E). However, in contrast to
1 in
1/
HK complexes (Fig. 4,
B and C), AL1 in AL1/
HK complexes
significantly increased its trypsin resistance in the presence of
K+/Mg2+ compared with that in the presence of
Mg2+ alone (Fig. 5, A and D). This
result may reflect the differences in the apparent K+
affinity of
1 and AL1 associated with
HK (Fig. 2).
Similar to AL1 associated with
HK, AL1 associated with
bl or
1 increased its trypsin resistance in the presence of
K+ or K+/Mg2+ compared with that in
the presence of Mg2+ alone (Fig. 5E). Because
1 but not
HK can support a stabilizing effect of
K+ on
1 in the presence of Mg2+,
but all
-subunits produce a stabilizing effect on AL1 under these
conditions, it may be suggested that
-isoforms have a less pronounced effect on the cation affinity of AL1 than on that of
1. This conclusion is also supported by the similar
apparent K+ affinity of AL1/
HK and AL1/
1
complexes (Fig. 2).
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Na+-dependent conformational changes
of AL1.
To provide further evidence for the Na+ transport capacity
of AL1- complexes, we next examined whether AL1/
complexes could undergo Na+-dependent conformational changes. The trypsin
resistance of AL1 in AL1/
bl and AL1/
1 complexes,
which are intrinsically more stable than AL1/
HK complexes, tended to
decrease in the presence of Na+ or
Na+/Mg2+ compared with that in the presence of
Mg2+ (Fig. 6B,
lanes 2-7, and Fig.
6D). On the other hand,
the trypsin resistance of AL1/
HK complexes rather increased when
Na+ was present alone or together with Mg2+/ATP
(Fig. 6A, lanes 3 and 7, and
Fig. 6C). However, this Na+ effect was
statistically not significant, probably due to the high intrinsic
trypsin sensitivity of AL1 in
HK complexes, which renders
quantifications difficult. Overall, these results support that AL1/
complexes interact with Na+ and respond with a
conformational change that is, however, more discreet than that induced
by K+.
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Ouabain-dependent conformational changes of AL1.
AL1- complexes are moderately sensitive to ouabain
(34). To verify whether this property is reflected in a
conformational change of AL1, we performed controlled proteolysis
assays on AL1/
HK, AL1/
bl, and AL1/
1 complexes in
ouabain/Na+/ATP or
ouabain/Mg2+/Tris-Pi conditions. Compared with
the control conditions (Na+/ATP,
Mg2+/Tris-Pi), ouabain had only slight or no
effect on the trypsin sensitivity of AL1/
complexes (data not
shown). A conformational effect of ouabain on AL1 was most apparent in
AL1/
bl complexes, in which ouabain decreased the trypsin resistance
of AL1 in the presence of Mg2+/Pi compared with
that in the presence of Mg2+/Tris-Pi alone
(Fig. 7, lanes 1-5). Under these conditions,
ouabain also modulated the production of distinct proteolytic fragments produced under control conditions and visible either in
immunoprecipitated (Fig. 7, lanes 2 and 3) or in
nonimmunoprecipitated (lanes 4 and 5) AL1
preparations. Thus, at least in AL1/
bl complexes, the moderate
ouabain sensitivity is reflected in a conformational change of AL1.
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DISCUSSION |
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The present study provides new information on two pending
questions concerning the structure-function relationship in nongastric H+-K+-ATPases, namely, on their ability to
transport Na+ and on the influence of different
-isoforms on their functional properties.
Na+ transport function of AL1.
It has recently been reported that rat colonic
H+-K+-ATPase (10) or human
AL1/HK complexes (17) expressed in Xenopus
oocytes or in HEK-293 cells, respectively, decrease
[Na+]i, suggesting that nongastric
X+-K+-ATPases may not only transport
H+ but also Na+ in exchange for K+.
On the other hand, measurements of the Na+ dependence of
ATPase activity of nongastric H+-K+-ATPases
have produced conflicting results. Whereas AL1-
HK complexes expressed in Sf21 insect cells exhibit an ATPase activity that is
inhibited rather than stimulated by Na+ (1), a
Na+-dependent K+-ATPase activity was
demonstrated in apical membranes of distal colon (8). In
this latter study, it can, however, not entirely be excluded that a
contaminating fraction of Na+-K+-ATPase in the
apical membrane preparation participates in the measured
Na+-dependent ATPase activity. In any case, both results,
activation or inhibition of ATPase activity, are consistent with
Na+ binding to nongastric
H+-K+-ATPases.
The functional role of -isoforms in AL1.
-Subunits of X+-K+-ATPases are necessary for
maturation of the enzymes and were shown to influence the intrinsic
transport properties of gastric H+-K+- and
Na+-K+-ATPases (13). Authentic
-subunits in gastric H+-K+-ATPase and in the
three Na+-K+-ATPase isozymes have been
identified and are represented by one gastric
H+-K+-ATPase and three
Na+-K+-ATPase
-isoforms. Significantly, for
nongastric H+-K+-ATPase, no specific
-subunit has been identified and the real subunit composition in
situ is still unknown. Indeed, both
Na+-K+-ATPase
1 (6,
29) and
3 (40) isoforms have been
suggested as the real partner subunit of nongastric
H+-K+-ATPase in native tissue based on
immunological evidence. These predictions are, however, not supported
by the observation that only
HK and
Na+-K+-ATPase
2-like isoforms,
but neither
1 nor
3, can act as efficient chaperones for the maturation of nongastric
H+-K+-ATPase
-subunits after expression in
different cells (2, 15, 39). On the other hand, in
agreement with Codina et al. (7), we observed in this
study that expression of a nongastric H+-K+-ATPase with
1-subunits
produces a number of functional pumps at the cell surface of
Xenopus oocytes similar to that produced after expression of
AL1 with gastric
HK, despite the poor chaperone activity of
1. This result indicates that, under conditions of marked overexpression as can be achieved in Xenopus oocytes,
a certain number of AL1/
1 complexes, which based on
their poor stability must be partially misfolded, can escape the
endoplasmic reticulum (ER) quality control that normally retains these
proteins in the ER. Because Xenopus oocytes have a limited
capacity for cell surface expression of endogenous and exogenous
proteins (3, 42), it may result that a similar number of
inefficiently assembled AL1/
1 and efficiently assembled
AL1/
HK become expressed at the cell surface. Thus the functional
expression of AL1/
1 complexes in oocytes cannot be used
as an argument to support
1 as the authentic subunit of
nongastric H+-K+-ATPases in situ.
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ACKNOWLEDGEMENTS |
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We thank Sophie Roy and Daniele Schaer for excellent technical
assistance. We thank G. Sachs for the cDNAs coding for rabbit gastric
H+-K+-ATPase - and
-subunits, K. Kawakami
for the cDNA coding for human Na+-K+-ATPase
1-subunit, and F. Jaisser for the cDNA coding for the
-subunit of
Bufo bladder.
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FOOTNOTES |
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This work was supported by Swiss National Fund for Scientific Research Grants 31-53721.98 and 31-64793.01 (to K. Geering) and National Heart, Lung, and Blood Institute Grant HL-36573 (to N. N. Modyanov).
Address for reprint requests and other correspondence: K. Geering, Institute Of Pharmacology and Toxicology, Rue du Bugnon 27, CH-1005 Lausanne, Switzerland (E-mail: kaethi.geering{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.
First published March 20, 2002;10.1152/ajpcell.00590.2001
Received 11 December 2001; accepted in final form 17 March 2002.
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