(Received for publication, January 19, 1996)
From the
The functional properties and the pharmacological profile of the
recently cloned cDNA colonic P-ATPase subunit (Crowson, M. S.,
and Shull, G. E.(1992) J. Biol. Chem. 267, 13740-13748)
were investigated by using the Xenopus oocyte expression
system. Xenopus oocytes were injected with
subunit cRNAs
from Bufo marinus bladder or rat distal colon and/or with
subunit cRNA from B. marinus bladder. Two days after
injection, K
uptake was measured by using
Rb
as a K
surrogate, and
pH measurements were performed by means of ion-selective
microelectrodes. Co-injection of
and
subunit cRNAs lead to
a large increase in
Rb
uptake, an
intracellular alkalinization, and an extracellular medium
acidification, as compared to
or
injection alone. These
results indicate that the colonic P-ATPase
subunit, like the
bladder
subunit, acts as a functional
H
,K
-ATPase, and that co-expression of
and
subunits is required for the function. External
K
activation of the
Rb
uptake had a K
440 µM for the bladder isoform (consistent with the previously reported
value (Jaisser, F., Horisberger, J. D., Geering, K., and Rossier, B.
C.(1933) J. Cell Biol. 123, 1421-1431)) and a K
730 µM for the colonic
isoform. Sch28080 was ineffective to reduce
Rb
uptake whereas ouabain inhibited the activity expressed from rat
colon
subunit with a K
of 970
µM when measured at the V
of the
enzyme. We conclude that, when expressed in Xenopus oocytes,
the rat colon P-ATPase
subunit encodes a ouabain-sensitive
H
,K
-ATPase.
Both colon and kidney are involved in chronic adaptation to
K homeostasis(1, 2) . Specific
transmembrane proteins in colonic and renal cells are responsible for
K
transport: whereas K
secretion is
mediated by diffusion through potassium channels, apical K
absorption occurs against an adverse electrochemical
transmembrane gradient, i.e. via active
process(es)(1) . Primary active K
transport
systems, requiring ATP hydrolysis, have been described both in colon
and kidney by physiological and biochemical
techniques(3, 4, 5, 6, 7) .
However, the molecules responsible for these colonic and renal
K
-ATPase activities are not yet defined.
Expression
of gastric H,K
-ATPase
and
subunit cRNAs has been reported recently all along the renal collecting
duct(8, 9) , but not in distal colon(10) . The
gastric H
,K
-ATPase may therefore
participate in K
handling in the kidney, and may
account for the ouabain-resistant, Sch28080-sensitive K
reabsorption process that has been described in this
tissue(3, 11, 12) . Recently, a novel P-type
ATPase
subunit cDNA has been cloned from a rat distal colon cDNA
library(10, 13) . It clearly belongs to a novel
subgroup of the
Na
,K
/H
,K
-ATPases
gene family and is equally related to, but distinct from, either the
Na
,K
-ATPase and the gastric
H
,K
-ATPase. In colon, the expression
of this P-ATPase is restricted to the most superficial cells of the
distal colon(13, 14) . In the kidney, it is not
expressed in rat maintained on a standard diet, but acute and chronic
K
deprivation markedly stimulates its mRNA expression
in the outer medullary collecting duct, but not in other nephron
segments(14) . Primary structure analysis suggests that this
novel P-ATPase
subunit may encode a K
- or
H
,K
-ATPase(10, 13) .
Its putative function, its restricted tissue- and cell-specific
expression, and its physiological regulation strongly suggests that it
corresponds to a
K
(H
,K
)-ATPase
involved in colon and renal K
reabsorption.
To test
this hypothesis, we have analyzed the properties of the colonic
P-ATPase subunit using a functional assay previously developed in
the Xenopus laevis oocyte for the expression of the toad
bladder H
,K
-ATPase(15) . We
demonstrate that this P-ATPase is indeed a ouabain-sensitive
H
,K
-ATPase, which may therefore be
involved in the ouabain-sensitive K
absorption
reported in both distal colon and the distal nephron.
Intracellular pH (pH) was calculated by the
relationship: pH
= pH
- (V
- V
)/S,
where pH
is the reference solution pH, V
the measured electrochemical potential difference for
H
, V
the transmembrane potential
difference, S the slope of the microelectrodes.
Extracellular pH measurements were performed at room temperature on individual oocytes incubated in a 1-µl droplet (surrounded by oil) of a weakly buffered solution, as described previously(15) . In some experiments, 1 mM ouabain was added to the external solution. The double-barrelled selective microelectrode was introduced into the droplet in order to measure the pH change of the solution. The conventional barrel was used as reference. Extracellular pH values reported here were obtained after 20 min of incubation.
Co-expression in Xenopus laevis oocytes of synthetic
cRNAs encoding the colonic P-ATPase subunit (10) and the
toad urinary bladder
subunit (19) leads to a large
increase in the uptake of
Rb
, a surrogate
of K
used as a tracer, when compared to oocytes
expressing the bladder
subunit alone as shown in Table 1.
Rb
uptake per oocyte is about twice that
measured under the same experimental conditions in Xenopus oocytes co-expressing the toad bladder
and
subunits (Table 1).
Rb
uptake in Xenopus oocytes injected with the colonic P-ATPase
subunit alone is
not different from those of oocytes injected with water (data not
shown). Thus, co-expression of the colonic
subunit together with
a
subunit is required for functional expression, as previously
reported for the
Na
,K
-ATPase(20) , the gastric
H
,K
-ATPase(17) , and the toad
bladder H
,K
-ATPase(15) .
Measurement of the intracellular pH of injected oocytes shows that
the co-expression of the colonic P-ATPase subunit with the toad
bladder
subunit induces an intracellular alkalinization, when
compared to oocytes expressing the
subunit alone;
similar findings were observed in oocytes expressing the bladder
H
,K
-ATPase (see Table 1), as
described previously(15) . On the other hand, a significant pH
decrease in an extracellular droplet surrounding the co-injected
oocytes is observed, whereas in the same conditions, extracellular pH
is not modified in the presence of oocytes expressing the
subunit alone (Table 1). Taken together, these results
indicate that, when co-expressed in Xenopus oocytes with the
subunit, the colonic P-ATPase
subunit acts as
a functional H
,K
-ATPase.
We next
evaluated the activation of the colonic
H,K
-ATPase by external
K
. Measurement of
Rb
uptake was performed using increasing concentrations of
extracellular K
, in oocytes expressing either the rat
colonic H
,K
-ATPase or the toad
urinary bladder H
,K
-ATPase
subunits, with the same
subunit, i.e. the bladder
subunit. K
for K
was estimated
to be
730 µM for the colonic
H
,K
-ATPase (Fig. 1); a K
440 µM for K
was found for the bladder
H
,K
-ATPase, a value similar to that
previously reported(15) . The K
for
K
of the colonic isoform is close to what has been
measured for the K
-activated ATPase activities in an
apical membrane preparation of rat distal colon by Del Castillo et
al.(6) (500-750 µM) and to the K
of K
absorptive fluxes measured
in the distal colon (520 µM)(7) . Interestingly,
the Hill coefficient determined for K
activation of
the colonic and bladder H
,K
-ATPases
is different (Hill coefficient:
,
0.98 ± 0.067, n = 4;
, 2.25 ± 0.39, n = 3; p < 0.01). A Hill coefficient of 2 for the
bladder H
,K
-ATPase strongly suggests
the presence of two or more binding sites with strong cooperativity.
Although a Hill coefficient of one, as observed for the rat colonic
H
,K
-ATPase, does not exclude the
presence of multiple binding sites, the very clear difference in Hill
coefficient between the two types of
H
,K
-pump suggests a difference of
stoichiometry, or at least a significant difference in the interaction
between binding sites. A stoichiometry of 2 K
/2
H
/1 ATP has been proposed for the gastric
H
,K
-pump(21) , but, at
present, nothing is known about this characteristic in the other
H
,K
pumps.
Figure 1:
K dose-dependent
activation of
Rb
uptake. Results are
normalized at 5 mM K
and expressed as mean
± S.E.; n = 24-40, from 3-4
independent experiments, depending on the K
concentration used. Curve-fitting is to a single model.
,
rat distal colon
/toad bladder
;
, toad bladder
/toad bladder
.
Functional expression
of the rat colonic H,K
-ATPase allows
us to analyze its pharmacological profile. In the rat distal colon,
K
absorption appears to be mediated by two different
apical Na
-independent K
-activated
ATPases(5, 6) . Their pharmacological characteristics
differ from those of both the
Na
,K
-ATPase and the gastric
H
,K
-ATPase. One colonic
K
-activated ATPase is sensitive to vanadate and to
ouabain but not to N-ethylmaleimide (inhibitor of V-type
H
-ATPase) or to omeprazole and Sch28080 (inhibitor of
the gastric H
,K
-ATPase), while the
other one is ouabain-insensitive but partly
Sch28080-sensitive(5, 6) . The rat distal colon
H
,K
-ATPase expressed in Xenopus oocytes is not sensitive to 500 µM Sch28080 (%
inhibition: 2.5 ± 9.5, n = 5), as compared to
the moderately sensitive bladder
H
,K
-ATPase (% inhibition: 41.6
± 3.4, n = 6). We next examined the effect of
ouabain on the rat colon H
,K
-ATPase
activity. Fig. 2shows that the colonic
H
,K
-ATPase studied in this paper is
sensitive to ouabain, with a K
of 970
µM, when the assay is performed at V
, in the presence of 5 mM K
. Extracellular acidification is also blocked by
1 mM ouabain (pH unit, 7.7 ± 0.1, n =
3, in the presence of ouabain, as compared to 5.8 ± 0.1, n = 9, in its absence). Since it is known that K
has a competitive effect on ouabain inhibition of the
Na
,K
-ATPase (22) and the toad
bladder H
,K
-ATPase(15) , we
evaluated the effect of extracellular K
concentration
on ouabain inhibition of
Rb
uptake.
Extracellular K
concentration affects ouabain affinity
since K
measured in the presence of 0.2 mM K
is about 70 µM, as compared to 970
µM in the presence of 5 mM K
(Fig. 3). It is concluded from the above experiments that
the colonic H
,K
-ATPase presently
expressed in Xenopus oocytes is ouabain-sensitive and
Sch28080-insensitive.
Figure 2:
Dose-dependent inhibition of Rb
uptake by ouabain. Results are
normalized to results obtained in the absence of ouabain and are
expressed as mean ± S.E.; n = 16-40, from
2-5 independent experiments, depending on the ouabain
concentration used.
, rat distal colon
/toad
bladder
;
, toad bladder
/toad bladder
.
Figure 3:
Dose-dependent inhibition of Rb
uptake by ouabain at two different
concentrations of extracellular K
, 0.2 mM (
) and 5 mM (
), in oocytes injected with rat
distal colon
/toad bladder
cRNAs. Results are normalized in the absence of inhibitor and are
expressed as mean ± S.E. One experiment was performed for the
dose-dependent inhibition of
Rb
uptake
performed in the presence of 0.2 mM K
using
8-10 oocytes per K
concentration. Data for the
dose-dependent inhibition of
Rb
uptake
performed in the presence of 0.55 mM K
are
those reported in Fig. 2.
The inhibition constant for ouabain measured
in the presence of 5 mM K differs from the
one reported in the literature for the distal colon ouabain-sensitive
K
-ATPase by Del Castillo et al.(6) .
In that paper, an inhibition constant of 100 µM was
reported when K
-activated ATPase activity was measured
in the presence of 20 mM KCl. As demonstrated above, the
ouabain affinity clearly depends on the extracellular K
concentration. Thus, the ouabain-sensitive
K
-ATPase measured by Del Castillo et al.(6) would be even more sensitive to ouabain if the
K
concentration was 5 mM instead of 20
mM. Several explanations for this discrepancy can be proposed.
First, different methodological procedures were used; however, we have
previously demonstrated that the determination of ouabain inhibition
kinetics in Xenopus oocyte is comparable to results obtained
with other techniques: the ouabain K
of B.
marinus Na
,K
-ATPase in a
purified enzyme preparation from the bladder(23) , in the TBM
cell line(20) , or in oocytes expressing the B. marinus
Na
,K
-ATPase subunits (20) was determined to be 100, 56, and 40 µM,
respectively.
Second, one should consider that the subunit
physiologically associated with the colonic
subunit may confer a
different pharmacological profile to the pump. The
subunit
associated in vivo with the colonic
subunit is not yet
defined. The gastric H
,K
-ATPase
subunit is not expressed in the colon(24) , while the
Na
,K
-ATPase
subunit
is(25) . Marxer et al.(25) reported that a
protein sharing common epitopes with the
Na
,K
-ATPase
subunit
was indeed present in the apical membrane of the rat distal
colon(25) . Whether this subunit corresponds to the
Na
,K
-ATPase
or to
an unidentified
subunit isoform remains unknown. It has been well
established that the
subunit affects the functional properties of
the Na
,K
-ATPase (26, 27) and, to some extent, those of the gastric
H
,K
-ATPase(28, 29) .
To date, the ouabain sensitivity of the diverse
heterodimers
already tested in Xenopus oocytes or in the insect Sf-9 cells
was not significantly affected by the type of
isoform used in the
assay(20, 30) . One exception is the rat gastric
subunit which slightly affects the K
for ouabain
binding from 10.1 to 5.8 nM, when associated with the sheep
Na
,K
-ATPase
subunit (27) .
A third possible explanation for the discrepancy
between the results of the current study and the previously reported
ouabain affinity of the colonic ouabain-sensitive
K-ATPase (6) would be that we are not dealing
with the same K
-ATPase. In fact, the so-called
ouabain-resistant K
-ATPase found in distal colon may
not be fully resistant to ouabain. The ouabain-resistant fraction of
the colonic K
-activated ATPase activities was
determined as the K
-ATPase activity resistant to 5
mM ouabain in the presence of 20 mM external
K
(6) or to 1 mM ouabain in the
presence of 15 mM external K
(5) .
This remaining activity could either correspond to a fully resistant
K
-ATPase or to the residual fraction of a moderately
sensitive K
-ATPase, as described here. Indeed, as
shown in Fig. 2, inhibition with 1 mM ouabain of the
colonic H
,K
-ATPase expressed in
oocytes resulted in only 50% inhibition when measured in the presence
of 5 mM external K
.
Interestingly, the
pharmacological profile described here, i.e. ouabain
sensitivity and Sch28080 insensitivity, is opposite to the one reported
for the expression of the same subunit cDNA in insect
cells(31) . These authors report on functional expression of
the colonic P-ATPase
subunit after infection of Sf9 cells with a
recombinant baculovirus. Sf9 infection induces a 3-fold increase in
K
-ATPase activity, measured as a Na
-
and Mg
-independent K
-activated
ATPase activity(31) . It should be noted that the
K
-ATPase activity was measured after expression of the
colonic
subunit alone, without
subunit. These data contrast
with what has been reported for the
Na
,K
-ATPase (32, 33) or the gastric
H
,K
-ATPase (34) in the same
functional expression system. In these cases, co-expression of
and
subunits was an absolute prerequisite for the induction of a
Na
,K
- or
H
,K
-ATPase
activity(32, 33, 34) . Thus, the
K
-activated ATPase activity reported after expression
of the colonic
subunit alone is difficult to reconcile with
previous studies. This ATPase activity may reflect the functional
expression of colonic
subunit oligomers, as reported recently by
Blanco et al.(35) for
Na
,K
-ATPase
/
oligomers, or
heterologous heterodimers formed with insect proteins, like insect
Na
,K
-ATPase
and/or
subunits. Such complexes may have specific functional and
pharmacological properties which may explain the differences with the
pharmacological characteristics reported here.
The colonic
H,K
-ATPase
subunit has been
reported recently to be expressed within the kidney when rats were
subjected to acute or chronic K
deprivation.
Expression was strictly restricted to the outer medullary collecting
duct cells and was not affected by aldosterone(14) . The role
of the ``colonic''
H
,K
-ATPase in renal K
handling and renal K
balance remains to be
defined. Rat distal nephron micropuncture
studies(36, 37) , as well as studies performed on
isolated perfused rat kidneys(38) , have shown that renal
K
absorption is inhibited by ouabain, suggesting that
``ouabain acts directly on distal potassium transport by
inhibiting active uptake of this ion at the luminal cell
membrane''(36) . The molecular basis underlying this
active K
transport has not been determined. Hayashi
and Katz (39) reported the presence of a ouabain-sensitive
K
-ATPase in the outer medullary collecting duct which
may explain the effect of ouabain on renal K
absorption. Altogether, the physiological data from the
literature, the functional results reported here, as well as the
cell-specific expression of the colonic
H
,K
-ATPase in the kidney, strongly
suggests that the ``colonic'' ouabain-sensitive
H
,K
-ATPase is involved in renal
K
handling during K
deprivation. In
rats maintained on a standard diet, a ouabain-resistant,
Sch28080-sensitive K
transport has been reported in
the cortical and medullary collecting ducts(3) . This
K
transport is probably mediated by a
ouabain-resistant, Sch28080-sensitive K
-ATPase present
in the same nephron segments (12) and may correspond to the
gastric H
,K
-ATPase
subunit
reported to be expressed all along the distal nephron in rats on a
standard diet(8) . Whether this ouabain-resistant K
reabsorption/K
-ATPase is indeed important for
renal K
conservation remains unclear. Dissection of
the specific role of the ouabain-sensitive and the ouabain-resistant
K
(H
,K
)-ATPases in
renal K
handling will require further experiments.
Genetically modified animals, such as knock-out mice having a null
mutation in the colonic H
,K
-ATPase
subunit gene and/or the gastric
H
,K
-ATPase
subunit gene, may be
useful to specifically address this question.