Functional expression of putative
H+-K+-ATPase
from guinea pig distal colon
Shinji
Asano1,
Satomi
Hoshina2,
Yumi
Nakaie2,
Toshiyuki
Watanabe3,
Michihiko
Sato4,
Yuichi
Suzuki5, and
Noriaki
Takeguchi2
1 Molecular Genetics Research
Center and 2 Faculty of
Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University,
Toyama 930-01; 3 Department of
Physiology and 4 Central
Laboratory for Research and Education, Yamagata University School of
Medicine, Yamagata 990-23; and
5 Laboratory of Physiology, School
of Food and Nutritional Sciences, University of Shizuoka, Shizuoka
422, Japan
 |
ABSTRACT |
A guinea pig cDNA
encoding the putative colonic
H+-K+-ATPase
-subunit (T. Watanabe, M. Sato, K. Kaneko, T. Suzuki, T. Yoshida, and Y. Suzuki; GenBank accession no. D21854) was functionally expressed in HEK-293, a human kidney cell line. The cDNA for the putative colonic
H+-K+-ATPase
was cotransfected with cDNA for either rabbit gastric H+-K+-ATPase
or Torpedo
Na+-K+-ATPase
-subunit. In both expressions,
Na+-independent,
K+-dependent ATPase
(K+-ATPase) activity was detected
in the membrane fraction of the cells, with a Michaelis-Menten constant
for K+ of 0.68 mM. The expressed
K+-ATPase activity was inhibited
by ouabain, with its IC50 value being 52 µM. However, the activity was resistant to Sch-28080, an
inhibitor specific for gastric
H+-K+-ATPase.
The ATPase was not functionally expressed in the absence of the
-subunits. Therefore, it is concluded that the cDNA encodes the
catalytic subunit (
-subunit) of the colonic
H+-K+-ATPase.
Although the
-subunit of the colonic
H+-K+-ATPase
has not been identified yet, both gastric
H+-K+-ATPase
and
Na+-K+-ATPase
-subunits were found to act as a surrogate for the colonic
-subunit for the functional expression of the ATPase. The present colonic
H+-K+-ATPase
first expressed in mammalian cells showed the highest ouabain
sensitivity in expressed colonic
H+-K+-ATPases
so far reported (rat colonic in
Xenopus oocytes had an IC50 = 0.4-1
mM; rat colonic in Sf9 cells had no ouabain sensitivity).
colonic proton-potassium-adenosinetriphosphatase; ouabain; proton
pump inhibitor
 |
INTRODUCTION |
THE TRANSEPITHELIAL
K+ transport in the colon is
involved in systemic K+
homeostasis. It was proposed that
K+ absorption in the distal colon
is an active process. Evidence is accumulating for the presence of a
colonic
H+-K+-ATPase,
which is localized in distal colon and mediates
K+ absorption and, possibly, acid
secretion in rat, rabbit, and guinea pig colons (1, 7, 20-24).
Suzuki and Kaneko (19, 20) reported that molecular mechanisms
responsible for ouabain-sensitive
K+ absorption and acid secretion
are localized in the middle and distal parts of the guinea pig colon.
Watanabe et al. (24) found ouabain-sensitive
K+-ATPase activity in the guinea
pig distal but not in the proximal colon. The activity was stimulated
by K+ and inhibited by vanadate,
responses similar to those of
Na+-K+-ATPase
and gastric
H+-K+-ATPase.
However, the colonic K+-ATPase
activity is not sensitive to Na+,
unlike
Na+-K+-ATPase
activity. Furthermore, the colonic
K+-ATPase is inhibited by ouabain
but not by omeprazole or
2-methyl-8-(phenylmethoxy)imidazo[1,2-
]pyridine-3-acetonitrile (Sch-28080), whereas gastric
H+-K+-ATPase
is inhibited by omeprazole and Sch-28080 but not by ouabain (23, 24).
Similarly, an ouabain-sensitive
K+-ATPase activity was observed in
the membrane fraction of rat colonic mucosae (7, 22).
Crowson and Shull (6) isolated from a rat colon library a novel cDNA
encoding a putative colonic
H+-K+-ATPase,
which is similar to gastric
H+-K+-ATPase
and
Na+-K+-ATPase
(60-65% amino acid identity). Recently, it was functionally expressed in Sf9 cells (16) and Xenopus
laevis oocytes (5), indicating that the putative rat
colonic
H+-K+-ATPase
encodes the catalytic subunit of colonic
H+-K+-ATPase.
Watanabe and colleagues cloned a novel cDNA (4242 bp) that encodes a
114-kDa product having 63% amino acid identity with
Na+-K+-ATPase
and 64% amino acid identity with gastric
H+-K+-ATPase
-subunits.1
It is likely that this is a homologue of rat colonic
H+-K+-ATPase
-subunit because of the 88% amino acid identity between them.
However, it has not been determined whether the molecular product of
this guinea pig cDNA shows
K+-ATPase activity. Here, we try
to functionally express the putative colonic
H+-K+-ATPase
cDNA in a HEK cell line as was previously done for gastric H+-K+-ATPase
(2, 3) and find that properties of the expressed ATPase were similar to
those reported for the membrane fraction of guinea pig distal colon,
that is, stimulated by K+,
sensitive to ouabain, and insensitive to Sch-28080.
 |
MATERIALS AND METHODS |
Materials.
HEK-293 cells (a human embryonic kidney cell line) were a kind gift
from Dr. Jonathan Lytton (Brigham and Women's Hospital, Harvard
Medical School, Boston, MA). pCIS2 vector was obtained from Genentech
(San Diego, CA). Restriction enzymes and other DNA and RNA modifying
enzymes were from Toyobo (Osaka, Japan), New England Biolabs, Life
Technologies, or Pharmacia Biotechnology (Tokyo, Japan). Sch-28080 was
obtained from Schering (Bloomfield, NJ). All other reagents were of
molecular biology grade or the highest grade of purity available.
cDNAs.
A cDNA encoding the putative colonic
H+-K+-ATPase
was cloned from guinea pig colonic cDNA library and subcloned in
pBluescript II SK(
). The sequence was registered in European
Molecular Biology Laboratories (EMBL) database (GenBank accession no.
D21854). cDNAs of gastric
H+-K+-ATPase
- and
-subunits were prepared from rabbit stomach as described
elsewhere (3). A cDNA of Torpedo
Na+-K+-ATPase
-subunit was a kind gift from Dr. Kawamura (University of
Occupational and Environmental Health, Kitakyushu, Japan). The cDNA of
the colonic
H+-K+-ATPase
was digested with Cla I and
Not I. The obtained fragment was
ligated into pCIS2 vector treated with
Cla I and
Not I.
Removal of 5'-noncoding sequence of the colonic
H+-K+-ATPase
cDNA.
The colonic
H+-K+-ATPase
cDNA between nucleotides
36 and 842 was amplified by PCR. The
PCR primers were 5'-GGCTCGAGCCCCGAGCCGCCCTCCAG-3' and
5'-CCAAGCTTGAAGCTATGCGTCCAATGATGG-3'. The 880-bp fragment was purified on the gel and digested with
Xho I and
BstE II. The cDNA cassette between
Xho I and
BstE II of the colonic
H+-K+-ATPase
cDNA was replaced by the PCR-derived fragment to remove the cDNA
sequence in its 5'-noncoding region (nucleotides
144 to
37).
Removal of 3'-noncoding sequence of the colonic
H+-K+-ATPase
cDNA.
The colonic
H+-K+-ATPase
cDNA was digested with Xho I and
Dra I. The 3,300-bp fragment was
purified on the gel and ligated with pBluescript SK(
) digested
with Xho I and
EcoR V to remove the cDNA sequence in
its 3'-noncoding region (nucleotides 3364-4101).
DNA sequencing.
For DNA sequencing, an Autoread DNA sequencing kit and an ALF-II DNA
sequencer (Pharmacia) were used.
Cell culture, transfection, and preparation of membrane fractions.
Cell culture of HEK-293 was carried out as described previously (3).
cDNA transfection was performed by the calcium phosphate method with 10 µg of cesium chloride-purified DNA per 10-cm dish. Cells were
harvested 2 days after the DNA transfection. Membrane fractions of HEK
cells were prepared as described previously (3). Briefly, cells in a
10-cm petri dish were washed with PBS and incubated in 2 ml of low
ionic salt buffer (0.5 mM MgCl2
and 10 mM Tris · HCl, pH 7.4) at 0°C for 10 min.
After the addition of phenylmethylsulfonyl fluoride (1 mM) and
aprotinin (0.09 U/ml), the cells were homogenized in a Dounce
homogenizer, and the homogenate was diluted with an equal volume of a
solution containing 500 mM sucrose and 10 mM
Tris · HCl, pH 7.4. The homogenized suspension was
centrifuged at 800 g for 10 min. The
supernatant was centrifuged at 100,000 g for 90 min, and the pellet was
suspended in a solution containing 250 mM sucrose and 5 mM
Tris · HCl, pH 7.4.
SDS-PAGE and immunoblot.
SDS-PAGE was carried out as described elsewhere (15). Membrane
preparations (30 µg of protein) were incubated in a sample buffer
containing 2% SDS, 2%
-mercaptoethanol, 10% glycerol, and 10 mM
Tris · HCl (pH 6.8) at room temperature for 2 min and applied to the SDS-polyacrylamide gel. Immunoblot was carried out as
described previously (3).
Antibody.
Antibody CHK-N was raised against the amino-terminal peptide (residues
10-24) of the guinea pig colonic
H+-K+-ATPase
-subunit (TKDTKQLGQEEGKKC). The amino acid sequence of the colonic
H+-K+-ATPase
-subunit in this segment is different from those of gastric H+-K+-ATPase
and
Na+-K+-ATPase
-subunits. The peptide was coupled with a carrier protein keyhole
limpet hemocyanin. The carrier- conjugated peptide was emulsified with
Freund's complete adjuvant, and the emulsion was injected
subcutaneously into Japanese White rabbits five times at intervals of
14-28 days.
Assay of
K+-ATPase
activity.
K+-ATPase activity was assayed in
1 ml of solution containing 50 µg of membrane protein, 3 mM
MgCl2, 3 mM ATP (Tris salt), 5 µM oligomycin, and 40 mM Tris · HCl (pH 7.4) in the
presence and absence of 15 mM KCl. After incubation at 37°C for 30 min, the inorganic phosphate released was measured as described
elsewhere (25). The K+-ATPase
activity was calculated as the difference between activities in the
presence and absence of KCl.
Protein was measured using a bicinchoninic acid protein assay kit from
Pierce (Rockford, IL), with BSA as a standard.
 |
RESULTS |
The protein deduced from the cDNA for the putative guinea pig colonic
H+-K+-ATPase
consists of 1,033 amino acids and has a molecular weight of 114,365. It
contains the putative phosphorylation site (Asp-385) and a
5'-FITC binding site (Lys-517), which are conserved in the members of P-type ATPases, and shows ~60% homology with gastric H+-K+-ATPase
and
Na+-K+-ATPase
-subunits. Therefore, it is likely that this protein is the
-subunit of colonic
H+-K+-ATPase.
Here, we transfected this putative guinea pig colonic H+-K+-ATPase
cDNA into HEK-293 cells after introduction into a mammalian expression
vector, pCIS2 (Genentech). The cDNA cloning of colonic H+-K+-ATPase
-subunit has not been reported. Therefore, this putative colonic
H+-K+-ATPase
cDNA was transfected without
-subunit cDNA or with
-subunit cDNA
of either rabbit gastric
H+-K+-ATPase
or Torpedo
Na+-K+-ATPase.
Figure 1 shows the Western
blot patterns of the HEK membrane fractions transfected with the
gastric
H+-K+-ATPase
-subunit cDNA alone or the colonic
H+-K+-ATPase
cDNA alone (full
) and fractions cotransfected with the colonic
H+-K+-ATPase
cDNA plus the gastric
H+-K+-ATPase
-subunit cDNA or Torpedo
Na+-K+-ATPase
-subunit cDNA (detected with antibody CHK-N raised against the
amino-terminal synthetic peptide of guinea pig colonic
H+-K+-ATPase).
When the cells were transfected with the colonic
H+-K+-ATPase
cDNA alone, a faint band was detected around 100 kDa. The intensity of
this band, that is, the expression of the colonic H+-K+-ATPase,
significantly increased when the cells were transfected together with
the gastric
H+-K+-ATPase
or
Na+-K+-ATPase
-subunit cDNA.

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Fig. 1.
Immunoblotting with CHK-N antibody of the membrane fraction of HEK
cells transfected with putative colonic
H+-K+-ATPase
cDNA. Thirty micrograms of HEK cell membrane fractions were applied to
the gel and blotted with antibody CHK-N directed against the putative
colonic
H+-K+-ATPase.
Lane 1, molecular weight standards;
lane 2, mock-transfected cells. Cells
were transfected with rabbit gastric
H+-K+-ATPase
-subunit cDNA (HK , lane 3),
putative colonic
H+-K+-ATPase
cDNA (full , lane 4), putative
colonic
H+-K+-ATPase
cDNA + rabbit gastric
H+-K+-ATPase
-subunit cDNA (lane 5), and
putative colonic
H+-K+-ATPase
cDNA + Torpedo
Na+-K+-ATPase
-subunit cDNA (NaK , lane
6).
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Next, we measured the K+-ATPase
activity of the membrane fractions of the transfected cells.
K+-ATPase activity was measured in
the absence of Na+ to eliminate
the possible effect of endogenous
Na+-K+-ATPase
present in the fraction. A small amount of
K+-ATPase activity was found in
the cells transfected with the colonic H+-K+-ATPase
cDNA alone (full
), which was slightly higher than that observed in
the cells transfected with the gastric
H+-K+-ATPase
-subunit cDNA alone. However, the cells cotransfected with the
colonic
H+-K+-ATPase
cDNA plus the gastric
H+-K+-ATPase
or
Na+-K+-ATPase
-subunit cDNA showed K+-ATPase
activity significantly higher than that found in the cells transfected
with the colonic
H+-K+-ATPase
cDNA alone (Fig. 2). This property of the
colonic
H+-K+-ATPase
was different from that of gastric
H+-K+-ATPase.
Gastric
H+-K+-ATPase
activity was found in the membrane fraction when the
H+-K+-ATPase
-subunit cDNA was cotransfected with the
H+-K+-ATPase
-subunit cDNA but not with the
Na+-K+-ATPase
-subunit cDNA (Fig. 3).

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Fig. 2.
K+-ATPase activities expressed in
transfected cells. K+-ATPase
activities of membrane fractions of cells transfected with rabbit
gastric
H+-K+-ATPase
-subunit cDNA, putative colonic
H+-K+-ATPase
cDNA, putative colonic
H+-K+-ATPase
cDNA + rabbit gastric
H+-K+-ATPase
-subunit cDNA, and putative colonic
H+-K+-ATPase
cDNA + Torpedo
Na+-K+-ATPase
-subunit cDNA were measured as described in
MATERIALS AND METHODS. Values are
means ± SE of 3 observations in 3 transfections.
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Fig. 3.
K+-ATPase activities expressed in
transfected cells. K+-ATPase
activities of membrane fractions of cells transfected with rabbit
gastric
H+-K+-ATPase
-subunit cDNA + Torpedo
Na+-K+-ATPase
-subunit cDNA (rab. H,K + Na,K ), rabbit gastric
H+-K+-ATPase
-subunit plus -subunit cDNAs (rab. H,K + rab. H,K ), and
rabbit gastric
H+-K+-ATPase
-subunit cDNA in the absence of -subunit cDNA were measured as
described in MATERIALS AND METHODS.
Values are means ± SE of 3 observations in 3 transfections.
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Furthermore, we modified the structures of the 5'- and
3'-untranslated regions of the colonic
H+-K+-ATPase
cDNA. Here, we prepared two different kinds of cDNA constructs of the
colonic
H+-K+-ATPase:
a cDNA truncated to its 5'-untranslated region (5'-
) and
cDNA truncated to its 3'-untranslated region (3'-
) (as
shown in MATERIALS AND METHODS), and
HEK cells were transfected with each of these cDNAs with the gastric
H+-K+-ATPase
-subunit cDNA. The expression level of
H+-K+-ATPase
was higher in the membrane fraction of the cells transfected with
5'-
colonic
H+-K+-ATPase
plus gastric
H+-K+-ATPase
-subunit cDNAs than that transfected with full-length colonic
H+-K+-ATPase
(full
) plus gastric
H+-K+-ATPase
-subunit cDNAs (Fig. 4). The expressed
K+-ATPase activity was also
significantly (~30%) higher in the cells transfected with
5'-
colonic
H+-K+-ATPase
plus gastric
H+-K+-ATPase
-subunit cDNAs (Fig. 5). It is likely
that some element suppresses the expression in the
5'-untranslated region of the colonic
H+-K+-ATPase
cDNA. It is noteworthy that similar findings were reported in the
expressions of urinary bladder
H+-K+-ATPase
and gastric
H+-K+-ATPase
(3, 12). Although it is difficult to estimate quantitative correlation
between the apparent level of expression and the measured level of
expressed K+-ATPase activity,
because Western analysis is semiquantitative, we observed as
reproducible experimental results in a qualitative manner that the
denser the ATPase band the higher the activity. Hereafter, we used the
5'-
construct as the colonic
H+-K+-ATPase
cDNA preparation and cotransfected this with the gastric H+-K+-ATPase
-subunit cDNA.

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Fig. 4.
Immunoblotting with CHK-N antibody of membrane fraction of HEK cells
cotransfected with modified colonic
H+-K+-ATPase
and gastric
H+-K+-ATPase
-subunit cDNAs. Thirty micrograms of HEK cell membrane fractions
were applied to the gel and blotted with antibody CHK-N directed
against the putative colonic
H+-K+-ATPase.
Lane 1, molecular mass standards;
lane 2, mock-transfected cells. Cells
were cotransfected with full-length colonic
H+-K+-ATPase
cDNA (full ) plus rabbit gastric
H+-K+-ATPase
-subunit cDNA (full + HK , lane
3), with colonic
H+-K+-ATPase
cDNA truncated in the 5'-untranslated region (5'- ) + rabbit gastric
H+-K+-ATPase
-subunit cDNA (lane 4), and with
colonic
H+-K+-ATPase
cDNA truncated in the 3'-untranslated region (3'- ) + rabbit gastric
H+-K+-ATPase
-subunit cDNA (lane 5).
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Fig. 5.
K+-ATPase activities expressed in
transfected cells. K+-ATPase
activities of membrane fractions of cells transfected with rabbit
gastric
H+-K+-ATPase
-subunit cDNA alone or with full-length colonic
H+-K+-ATPase
cDNA (full ) alone or cotransfected with modified colonic
H+-K+-ATPase
(full , 5'- , and 3'- ) and gastric
H+-K+-ATPase
-subunit cDNAs were measured as described in
MATERIALS AND METHODS. Values are
means ± SE of 3 observations in 3 transfections.
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The K+-ATPase activity was almost
completely inhibited by 2 mM ouabain but not inhibited by 100 µM
Sch-28080 (only 8% inhibition with 100 µM Sch-28080), which is an
inhibitor specific to gastric H+-K+-ATPase
(18). These properties are qualitatively similar to those of
K+-ATPase found in guinea pig
distal colon (23, 24). The inhibitory effects of ouabain on the
K+-ATPase expressed in HEK cells
are quantitatively shown in Fig. 6. Ouabain
inhibited the K+-ATPase in a
concentration-dependent manner, with an
IC50 of ~52 µM. This value was
smaller than that reported for the rat colonic H+-K+-ATPase
expressed in Xenopus oocytes
[inhibition constant
(Ki) value of
970 µM] (5) but higher than that reported for guinea pig
colonic epithelial cells
(Ki value of 3.2 µM) (24). Figure 7 shows the effects of
K+ concentrations on the
K+-ATPase expressed in the
membrane fraction of cells transfected with the colonic
H+-K+-ATPase
and gastric
H+-K+-ATPase
-subunit cDNAs. The ATPase activity was stimulated with K+ in a concentration-dependent
manner; the Michaelis-Menten constant (Km) value was
0.68 mM. This value was close to the value reported for the rat colonic
H+-K+-ATPase
expressed in Xenopus oocytes
(Km value of 730 µM) (5) but larger than that reported for the gastric
H+-K+-ATPase
expressed in HEK cells
(Km value of 0.2 mM) (3) and much higher than that reported for guinea pig colonic
epithelial cells
(Km value of 55 µM) (24).

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Fig. 6.
Effects of ouabain concentrations on expressed colonic
K+-ATPase activity.
K+-ATPase activities of the
membrane fraction of the HEK cells cotransfected with the putative
colonic
H+-K+-ATPase
-subunit (5'- ) and rabbit gastric
H+-K+-ATPase
-subunit cDNAs were measured as a function of ouabain
concentrations. K+-ATPase
activities are expressed as percentage of control values measured in
the absence of ouabain. Values are means ± SE of 3 observations in
3 transfections. K+-ATPase
activity in the absence of ouabain was 0.62 ± 0.09 µmol · mg 1 · h 1.
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Fig. 7.
Effects of K+ concentrations on
expressed colonic K+-ATPase
activity. K+-ATPase activities of
the membrane fraction of HEK cells cotransfected with the putative
colonic
H+-K+-ATPase
-subunit (5'- ) and rabbit gastric
H+-K+-ATPase
-subunit cDNAs were measured as a function of
K+ concentrations.
K+-ATPase activity was calculated
as the difference between the ATPase activities in the presence and
absence of KCl. Values are means ± SE of transfections.
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DISCUSSION |
The presence of an active K+
transporter and proton pump in the apical membrane of distal colonic
cells has been postulated in many species, including rabbit, rat, and
guinea pig. Gustin and Goodman (9, 10) first reported that an
ouabain-resistant K+-ATPase is
present that is inhibited by vanadate and forms a phosphorylated intermediate in rabbit descending colon. Subsequently, a
K+-dependent proton pump activity
was observed in the membrane vesicles from rabbit distal colon (14).
Similar K+-ATPase activities were
also observed in the membrane fractions of guinea pig (21, 23, 24) and
rat distal colons (7, 22). The former was sensitive to ouabain and
insensitive to Sch-28080, and the latter was partly inhibited by
ouabain and Sch-28080. These findings suggest that in the distal colon
there should be an isoform of gastric
H+-K+-ATPase
that is involved in acid secretion and
K+ absorption. A putative colonic
H+-K+-ATPase
was first cloned from rat distal colon (6). It has 60-65% amino
acid identity with
Na+-K+-ATPase
and gastric
H+-K+-ATPase
-subunits. Recently, the rat colonic cDNA without exogenous
-subunit cDNA of
Na+-K+-ATPase
or
H+-K+-ATPase
was functionally expressed in insect Sf9 cells (16). Successively, the
rat colonic
H+-K+-ATPase
was expressed in Xenopus oocytes;
oocytes were injected with the rat colonic
H+-K+-ATPase
cRNA together with the toad bladder
-subunit
H+-K+-ATPase
cRNA (13). The oocytes showed
Rb+ uptake, intracellular
alkalinization, and acidification of the extracellular medium (5).
Therefore, it is concluded that the putative rat colonic
H+-K+-ATPase
cDNA encodes an
-subunit of colonic
H+-K+-ATPase.
Another cDNA cloned from guinea pig distal colon by Watanabe et al.
(GenBank accession no. D21854) may be regarded as a putative colonic
H+-K+-ATPase
because this cDNA clone shows 88% amino acid identity with the rat
colonic
H+-K+-ATPase
cDNA. In the present study, we expressed the putative
-subunit of
guinea pig colonic
H+-K+-ATPase
together with the
-subunit of rabbit gastric
H+-K+-ATPase
or Torpedo
Na+-K+-ATPase
and examined the properties of the expressed ATPases. When the cells
were transfected with the putative colonic
H+-K+-ATPase
cDNA in combination with the rabbit gastric
H+-K+-ATPase
-subunit cDNA, K+-ATPase
activity was found in the membrane fraction of the cells. This
K+-ATPase activity in HEK cells
was inhibited by ouabain but not by Sch-28080. Therefore, it is
regarded that this putative colonic H+-K+-ATPase
cDNA encodes an
-subunit of guinea pig colonic
H+-K+-ATPase.
The K+-ATPase activity expressed
in HEK cells showed lower sensitivity to ouabain and lower affinity for
K+ than that in the membrane
fraction of guinea pig colonic mucosae (Ki values for
ouabain of 52 µM and 3.2 µM and
Km values for
K+ of 0.68 mM and 55 µM,
respectively). One explanation for this discrepancy is that the gastric
-subunit associated with the colonic
H+-K+-ATPase
-subunit may have conferred a different sensitivity to inhibitors
and different affinity for cations. The colonic
H+-K+-ATPase
-subunit is likely to be assembled with its endogenous
-subunit
in the colonic mucosae, resulting in high sensitivity to ouabain and
high affinity for K+. However, the
-subunit associated in vivo with the colonic
-subunit has not
been cloned yet. Difference in
-subunits may result in the
conformational difference in expressed
H+-K+-ATPases.
Similar findings were reported for the expression of rat colonic
H+-K+-ATPase.
The rat colonic
H+-K+-ATPase
expressed in Xenopus oocytes by
coinjection of the colonic
-subunit and toad bladder
-subunit
cRNAs was sensitive to ouabain (Ki = 0.97 mM)
and insensitive to Sch-28080 (5). Furthermore, when expressed with the
1-subunit of
Na+-K+-ATPase
or gastric
H+-K+-ATPase
-subunit in oocytes, the Rb+
uptake was sensitive to ouabain
(IC50 = 0.4-0.6 mM) and
insensitive to Sch-28080 (4). These sensitivities of the expressed
ATPase to ouabain were lower than that reported for the rat colonic
mucosae (Ki = 0.1 mM) (7). In addition, when the same rat colonic
H+-K+-ATPase
-subunit cDNA was introduced in insect Sf9 cells without
-subunit
cDNAs, the expressed
H+-K+-ATPase
activity was insensitive to ouabain (1 mM) and slightly inhibited by
Sch-28080 (18% inhibition by 100 µM Sch-28080) (16).
It is very interesting that the colonic
H+-K+-ATPase
cDNA product can be functionally assembled with both the cDNA product
of gastric
H+-K+-ATPase
-subunit and that of the
Na+-K+-ATPase
-subunit. A similar finding was reported for the functional expression of rat colonic
H+-K+-ATPase
in Xenopus oocytes, in which rat
colonic
H+-K+-ATPase
-subunit assembled with either the rat gastric
H+-K+-ATPase
-subunit or the rat
Na+-K+-ATPase
-subunit, resulting in
86Rb+
uptake in both cases (4). The
Na+-K+-ATPase
-subunit can assemble not only with the
Na+-K+-ATPase
-subunit but also with the
H+-K+-ATPase
-subunit, resulting in a functional heteroligomer (11). On the other
hand, the functional expression of
H+-K+-ATPase
using the gastric
H+-K+-ATPase
-subunit in combination with the
Na+-K+-ATPase
-subunit has not been reported. In our expression system using HEK
cells presented here, K+-ATPase
activity was not functionally expressed when the cells were
cotransfected with gastric
H+-K+-ATPase
-subunit and
Na+-K+-ATPase
-subunit cDNAs (Fig. 3). These findings suggest that the
colonic
H+-K+-ATPase
-subunit is intermediary between
Na+-K+-ATPase
and
H+-K+-ATPase
-subunits.
The protein encoded in the human
ATP1AL1 gene is another member of
nongastric
H+-K+-ATPase
that shares 86-89% amino acid identity with rat and guinea pig
colonic
H+-K+-ATPases.
Recently, Modyanov et al. (17) reported the functional expression of
ATP1AL1 cRNA product in
Xenopus oocytes. Oocytes coinjected
with the ATP1AL1 cRNA and that of
rabbit gastric
H+-K+-ATPase
-subunit showed
86Rb+
uptake. This uptake was sensitive to ouabain
(Ki = 13 µM)
and almost insensitive to Sch-28080 (17). Successively, Grishin et al.
(8) reported the functional expression of this cDNA in HEK cells. The
cells cotransfected with cDNAs for
ATP1AL1 and rabbit gastric
H+-K+-ATPase
-subunit also showed
86Rb+
uptake. The uptake was inhibited by ouabain
(Ki = 42 µM)
and Sch-28080
(Ki = 131
µM). Thus the functional properties of
ATP1AL1 gene product are similar to
those exhibited by rat and guinea pig colonic
H+-K+-ATPases.
It is not clear, however, whether
ATP1AL1 is expressed in the colon.
In conclusion, the protein encoded by the putative colonic
H+-K+-ATPase
cDNA exhibited an ouabain-sensitive, Sch-28080-insensitive K+-ATPase in combination with the
expression of the gastric
H+-K+-ATPase
or
Na+-K+-ATPase
-subunits.
 |
ACKNOWLEDGEMENTS |
This study was supported in part by a Grant-in-Aid for Encouragement of
Young Scientists (to S. Asano) and Scientific Research on Priority
Areas (to S. Asano, Y. Suzuki, and N. Takeguchi) from the Ministry of
Education, Sports, Science, and Culture in Japan.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
1
T. Watanabe, M. Sato, K. Kaneko, T. Suzuki, T. Yoshida, and Y. Suzuki; GenBank accession no. D21854.
Address for reprint requests: S. Asano, Molecular Genetics Research
Center, Toyama Medical and Pharmaceutical Univ., 2630 Sugitani, Toyama,
930-01, Japan.
Received 26 January 1998; accepted in final form 12 May 1998.
 |
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