Departments of Internal Medicine and Pediatrics, Yale University, New Haven, Connecticut 06520-8019
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ABSTRACT |
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Active K absorption in the rat distal colon is energized
by an apical H-K-ATPase, a member of the gene family of P-type ATPases. The H-K-ATPase -subunit (HKc
) has been cloned and characterized (together with the
-subunit of either Na-K-ATPase or gastric H-K-ATPase) in Xenopus oocytes as ouabain-sensitive
86Rb uptake. In contrast, HKc
, when expressed in Sf9
cells without a
-subunit, yielded evidence of ouabain-insensitive
H-K-ATPase. Because a
-subunit (HKc
) has recently been cloned
from rat colon, this present study was initiated to determine whether
H-K-ATPase and its sensitivity to ouabain are expressed when these two
subunits (HKc
and HKc
) are transfected into a mammalian cell
expression system. Transfection of HEK-293 cells with HKc
and HKc
cDNAs resulted in the expression of HKc
and HKc
proteins and
their delivery to plasma membranes. H-K-ATPase activity was identified in crude plasma membranes prepared from transfected cells and was
1) saturable as a function of increasing K concentration with a
Km for K of 0.63 mM; 2) inhibited by
orthovanadate; and 3) insensitive to both ouabain and
Sch-28080. In parallel transfection studies with HKc
and Na-K-ATPase
1 cDNAs and with HKc
cDNA alone, there was expression of
ouabain-insensitive H-K-ATPase activity that was 60% and 21% of that
in HKc
/HKc
cDNA transfected cells, respectively. Ouabain-insensitive 86Rb uptake was also identified in
cells transfected with HKc
and HKc
cDNAs. These studies establish
that HKc
cDNA with HKc
cDNA express ouabain-insensitive
H-K-ATPase similar to that identified in rat distal colon.
potassium absorption; epithelial transport; rat colon; rubidium-86 uptake; hydrogen-potassium-adenosinetriphosphatase
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INTRODUCTION |
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ACTIVE POTASSIUM ABSORPTION in mammalian distal colon
is mediated by one or more H-K-ATPases of the P-type gene family of ion
transport ATPases (2, 10, 16, 30). P2-type ATPases are
heterodimers that consist of - and
-subunits, whereas
P1-type ATPases only consist of
-subunits. Colonic
H-K-ATPase
-subunit (HKc
) (8) is localized in apical membranes of
surface epithelial cells (15, 24), and its mRNA and protein expression
are increased threefold in the distal colons of dietary Na-depleted
rats (24). Functional expression of HKc
cDNA without
-subunit in
Sf9 cells revealed H-K-ATPase activity that was insensitive to ouabain
(18). In contrast, HKc
cRNA when coexpressed with the
-subunit of Na-K-ATPase (NaK
1) or gastric H-K-ATPase (HKg
) either in
Xenopus oocytes or in human embryonic kidney 293 cells (HEK-293
cells) demonstrated ouabain-sensitive 86Rb uptake (5, 7).
Neither of these studies reported the expression of K-dependent ATP
hydrolysis (i.e., H-K-ATPase activity) (5, 7).
Recently, we have isolated and identified a -subunit from rat distal
colon that we refer to as HKc
and that is expressed in both apical
and basolateral membranes of rat distal colon (23). We have also
demonstrated by coimmunoprecipitation the physical association of this
HKc
protein with HKc
protein and its upregulation in apical
membrane by dietary K depletion (23). As a result, we proposed that
this is the
-subunit for the colonic H-K-ATPase. This present study
was, therefore, designed to determine whether HKc
, when coexpressed
with HKc
cDNA in mammalian cells, exhibited ATPase activity. In this
present communication, we report that both HKc
and NaK
1 proteins
formed a functional enzyme complex with HKc
protein in HEK-293 cells
and yielded the expression of both ouabain-insensitive H-K-ATPase
activity and 86Rb uptake.
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MATERIALS AND METHODS |
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HEK-293 cells were a gift from Dr. B. Forbush (Yale University). pcDNA 3.1+ vector was purchased from Invitrogen (Carlsbad, CA); restriction enzymes were from New England Biolabs (Beverly, MA). All other reagents were of molecular biology or analytical grade.
Plasmid construction.
The full-length cDNAs encoding the rat colonic HKc (8), the colonic
HKc
(23), and the rat NaK
1 (20) were modified by PCR both at the
5'end and the 3'end with BamH I and EcoR I, respectively. The HKc
, HKc
, and NaK
1 cDNAs were digested
separately with BamH I and EcoR I enzymes and ligated
using T4 DNA ligase independently into pcDNA 3.1+ that had been
previously digested with BamH I and EcoR I and
dephosphorylated with calf intestinal phosphatase. The ligation mixture
was transformed into XL1-blue Escherichia coli cells, and the
plasmid DNAs were prepared according to the method described by Morelle
(21). The plasmid DNAs were digested with BamH I and
EcoR I enzymes to release the insert, and the plasmids
containing expected size inserts were sequenced by an automated
fluorescence sequencer (William Keck Sequencing Facility, Yale
University). Plasmids containing full-length cDNAs (hereafter referred
to as pHKc
, pHKc
, and pNaK
1) with the correct sequences were
used for the expression studies.
Cell culture and transfection.
COS-7 cells were grown (13) in high-glucose DMEM containing 10% fetal
bovine serum, 50 U/ml penicillin, 50 µg/ml streptomycin, and 2 mM
L-glutamine (complete DMEM). Transfections were carried out
in subconfluent COS-7 cells with vector alone, pHKc alone, pHKc
alone, pNaK
1 alone, pHKc
/pHKc
, or pHKc
/pNaK
1 in
independent transfections using superfect transfection reagent (Qiagen,
Chatsworth, CA) according to the manufacturer's recommendations. In
brief, plasmid DNA was mixed with serum, antibiotic-free DMEM, and the recommended amount of superfect reagent and incubated at room temperature for 30 min. After incubation, 2 ml of serum and
antibiotic-free DMEM were added. The entire mixture was added to the
cells that previously had been washed three times with serum and
antibiotic-free medium. The plates were incubated at 37°C in a
humidified CO2 incubator for 2 h. The medium was removed
and washed with 5 ml medium. Finally, 6 ml of medium were added, and
the cells were placed in the CO2 incubator at 37°C for
60 h. After 60 h, the medium was removed, the cells were washed with
1× PBS three times, 1× PBS was added, and the cells were
scraped and harvested by centrifugation.
Plasma membrane preparation.
Cells were harvested and washed twice with 1× PBS. Cells were
then resuspended in sonication buffer containing 50 mM
Tris · HCl (pH 7.4), 1 mM EDTA, 250 mM sucrose, and 1 mM phenylmethylsufonyl fluoride. The cells were then sonicated for 15 s
each time (3 times) with 30-s intervals between them. The lysate was
centrifuged at 30,000 g for 30 min at 4°C (Sorvall RC5B,
SS34 rotor), and the membrane pellet was resuspended in a buffer
containing 50 mM Tris · HCl (pH 7.4) and 250 mM
sucrose. These crude plasma membranes were used for enzyme assay and
Western blot analysis. Protein was estimated by the Bradford method
using bovine -globulin as standard (3).
Assay of ATPases. H-K-ATPase activity was assayed in 0.5 ml of reaction volume containing 50 µg of membrane protein, 40 mM Tris · HCl (pH 7.4), 3 mM MgCl2, and 10 µM ouabain in the presence and absence of 5 mM KCl. The reaction was initiated by the addition of 3 mM ATP (Tris salt) and incubated at 37°C for 1 h; inorganic phosphate released was measured, as described previously (9). H-K-ATPase activity was calculated as the difference between activities in the presence and absence of KCl. Na-K-ATPase activity was also assayed using the above reaction condition but also included 100 mM NaCl and was calculated as the difference between activities in the presence and absence of Na. The specific activity of the enzyme is expressed as nanomoles of phosphorus liberated per milligram of protein per minute.
SDS-PAGE and Western blot analysis.
SDS-PAGE was carried out, as previously described (17). Fifty
micrograms of membrane protein were incubated in a sample buffer
containing 10 mM Tris · HCl (pH 6.8), 2% SDS, 2%
mercaptoethanol, and 10% glycerol at room temperature for 5 min and
loaded on an SDS-polyacrylamide gel. Western blot analyses were
performed using HKc, HKc
, and NaK
1 antibodies, as previously
described (23).
Immunofluorescence studies.
Stably transfected HEK-293 cells that express HKc protein alone and
express both HKc
and HKc
proteins were grown on 22 × 22 mm
glass coverslips for 48 h. The cells were washed with 1× PBS,
fixed for 20 min in 4% paraformaldehyde prepared in 1× PBS, and
processed as described previously (12). The fixed cells were incubated
with appropriate antibodies for 1 h at room temperature at a dilution
of 1:100. CY3-conjugated anti-rabbit IgG (Amersham) secondary
antibodies were used at a dilution of 1:2,000 at room temperature for 1 h. Immunofluorescence images were visualized using a Zeiss-Axiophot microscope.
86Rb uptake studies. Tissue culture plates (24 wells) were treated with 0.1% (wt/vol) poly-L-lysine (200 µl/well) for 5 min in a tissue culture Laminar flow hood. The plates were then washed with sterilized deionized water and were allowed to dry for 5 min. For the untransfected cells, 1 × 105 cells/well were plated and grown in growth media (low-glucose DMEM containing 10% fetal bovine serum and 50 units penicillin/streptomycin solution). The transfected cell lines were grown in the same growth media but with the addition of 900 µg/ml G418. The cells grew at a slower rate but did not wash off during washing while performing 86Rb uptake studies in the poly-L-lysine-treated plates compared with those grown in the untreated plates.
The cells were grown for 4-5 days and fresh media were changed every 48 h. After the cells had grown to complete confluency (about 5 days), the media were aspirated and the cells were washed six times with 500 µl uptake buffer containing 145 mM NaCl, 1 mM KCl, 10 mM glucose, 1.2 mM MgCl2, 1.0 mM CaCl2, 2 mM NaH2PO4, 32 mM HEPES, and 200 µM bumetanide, pH 7.4. Bumetanide was added to block any 86Rb uptake by Na-K-2Cl cotransporter. Preliminary studies had shown that 86Rb uptake was linear for up to 30 min in both the untransfected and the transfected cell lines; therefore, all uptake studies were performed for 10 min. Additional preliminary studies in the untransfected cell line demonstrated that 10 µM ouabain reduced 86Rb uptake by ~95%, which was attributed to uptake via Na-K-ATPase; therefore, all studies were performed in the presence of 10 µM ouabain. The cells were initially incubated for 20 min at 37°C in uptake buffer solution. The uptake buffer solution was then replaced by 200 µl uptake buffer containing 86Rb (4 µCi/ml) in the presence or absence of either 1 mM ouabain or 100 µM vanadate, and the cells were incubated for 10 min at 37°C. After incubation and removal of buffer, the cells were washed six times with 500 µl ice-cold stop buffer (10 mM HEPES-Tris, 100 mM MgCl2, pH 7.4). The cells were solubilized in 500 µl of 2% SDS/0.1 N NaOH, and 10 µl of the solubilized cell lysate were used to estimate the protein concentration by the method of Bradford (3). The remaining cell lysate was mixed with 3.5 ml scintillation solution, and radioactivity was measured in a scintillation photometer. 86Rb uptake was expressed as nanomoles per milligrams of protein per 10 min. ![]() |
RESULTS |
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To establish that all the constructs expressed the expected
size proteins, we first transiently transfected COS-7
cells using the plasmid containing full-length HKc, HKc
, and
NaK
1 cDNAs as either individual plasmid constructs (pHKc
,
pHKc
, pNaK
1) or as a combination of two plasmids
(pHKc
/pHKc
or pHKc
/pNaK
1). The results of these
expression studies revealed that untransfected COS-7 cells or the cells
transfected with vector alone did not express either HKc
or HKc
proteins (Fig. 1, A and B,
lanes 1-3). Cells transfected with pHKc
alone, pHKc
/pHKc
, or pHKc
/pNaK
1 expressed HKc
protein, as detected by HKc
antibody (Fig. 1A, lanes 4, 7, and 8, respectively). Cells transfected with pHKc
alone or pHKc
/pHKc
expressed HKc
protein, as detected by
HKc
antibody (Fig. 1B, lanes 5 and 7).
Cotransfection of pHKc
/pHKc
resulted in the expression of less
HKc
protein compared with that identified in cells transfected with
pHKc
alone. In contrast, HKc
protein was expressed in higher
amounts than HKc
protein whether pHKc
was transfected alone or
cotransfected with pHKc
. Several attempts were made to alter the
ratio of the
/
constructs for transfections to result in the
expression of approximately equal amounts of HKc
and HKc
proteins. None of these approaches resulted in the expression of
H-K-ATPase activity in cell membranes from these transfected cells.
Therefore, we proceeded to establish stable cell lines that would
express HKc
and HKc
protein in approximately equal amounts.
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Several stable HEK-293 cell lines were developed after transfection
with the following cDNAs: 1) both HKc and HKc
, 2)
HKc
and NaK
1, 3) HKc
alone, and 4) HKc
alone. Western blot analyses were performed using the crude membranes
from the stable cell lines (Fig. 2). The
untransfected HEK-293 cells did not express either HKc
protein or
HKc
protein. Cell lines stably transfected with pHKc
alone,
pHKc
/pHKc
, or pHKc
/pNaK
1 expressed HKc
protein, as
detected by HKc
antibody (Fig. 2A, lanes 3, 5, and 6, respectively). Cell lines stably transfected with
pHKc
alone or with pHKc
/pHKc
expressed HKc
protein, as
detected by HKc
antibody (Fig. 2B, lanes 4 and
5), whereas those cell lines transfected with pHKc
/pNaK
1
expressed NaK
1 as protein, as detected by NaK
1 antibody (Fig.
2C, lane 6).
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To establish whether the expressed HKc or HKc
proteins were
transported to plasma membranes, immunofluorescence studies were
performed. The results of such immunofluorescence studies are presented
in Fig. 3. The stable cell line expressing
both HKc
and HKc
proteins stained with HKc
antibody (Fig.
3C) and stained with HKc
antibody (Fig. 3D)
demonstrate that both HKc
and HKc
proteins are transported to and
localized in the plasma membranes. Staining (Fig. 3A) was not
identified in the same stable cell line expressing both HKc
and
HKc
proteins when stained with preimmune serum, indicating that the
staining produced by HKc
and HKc
antibodies is specific (Fig.
3A). HKc
protein in the pHKc
alone stably transfected
cell line was not efficiently localized to the plasma membrane (Fig.
3B). Therefore, HKc
protein requires a
-subunit to be
transported and localized in the plasma membrane.
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ATPase activity was determined in the untransfected HEK-293 cell
membranes before establishing the assay conditions for the transfected
cells. Only Na-K-ATPase activity was identified in the untransfected
HEK-293 cell membranes (15.8 ± 1.3 nmol Pi
liberated · mg
protein-1 · min-1). This
endogenous Na-K-ATPase activity was completely inhibited by 10 µM
ouabain (data not presented). Similar levels of Na-K-ATPase activities
(16.2 ± 0.9 and 16.1 ± 0.8 nmol Pi
liberated · mg
protein-1 · min-1) were
identified in the two doubly transfected cell lines, pHKc/pHKc
and pHKc
/NaK
1, respectively. Although Na-K-ATPase activity was present endogenously in the untransfected cell line, increased levels of Na-K-ATPase were not expressed in the cell lines transfected with both
- and
-subunits. Thus all subsequent assays included 10 µM ouabain in the ATPase assay in membranes from transfected cells.
To determine whether H-K-ATPase is expressed in the stable cell lines,
H-K-ATPase assay was determined in membranes prepared from the
different cell lines. The maximal H-K-ATPase activity was observed in
the pHKc/pHKc
stably transfected cell line (Fig. 4). In contrast, H-K-ATPase
activity in the pHKc
/pNaK
1 stably transfected cell line was
60% of that in the pHKc
/pHKc
stably transfected cell line.
Cells that had been stably transfected with pHKc
alone expressed
H-K-ATPase activity that was 21% of maximal activity. Minimal
H-K-ATPase activity was noted both in cells that had been stably
transfected with pHKc
alone and in the untransfected cell lines.
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The effect of K+ concentrations on the H-K-ATPase activity
in the crude plasma membranes prepared from the cell line stably transfected with pHKc/pHKc
was also studied to determine
whether the in vitro expressed H-K-ATPAse activity in plasma membranes of HEK-293 cells had similar or different properties from those of
H-K-ATPase activity in native colonic apical membranes. Increasing K
concentrations in the incubation medium stimulated and saturated H-K-ATPase activity (Fig. 5).
Analysis of this data with a Linweaver-Burk plot yielded a
Km for K of 0.63 mM. This kinetic constant is
comparable to those previously reported for H-K-ATPase in native rat
colonic apical membranes (Km = 0.75 mM) (9), the
rat colonic H-K-ATPase expressed as 86Rb uptake in
Xenopus oocytes (Km = 0.73 mM) (7), and the
Km calculated for rat H-K-ATPase
-subunit
expressed in Sf9 cells (Km = 1.2 mM) (18).
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To establish the functional properties of the H-K-ATPase activity
identified in membranes from cell lines stably transfected with
pHKc/pHKc
or pHKc
/pNaK
1, the effect of different ATPase inhibitors on H-K-ATPase activity was determined. One millimolar orthovanadate inhibited H-K-ATPase activity by ~75%. In contrast, neither 1 mM ouabain nor 0.5 mM Sch-28080 altered H-K-ATPase activity (Fig. 6).
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86Rb uptake was performed in untransfected, in
pHKc/pHKc
transfected, and in pHKc
/pNaK
1 transfected
HEK-293 cell lines (Fig. 7.) Preliminary
studies had shown that 86Rb uptake was linear for up to 30 min in both the untransfected and the transfected cell lines;
therefore, all uptake studies were performed for 10 min. Additional
preliminary studies in the untransfected cell line demonstrated that 10 µM ouabain reduced 86Rb uptake by ~95%, which was
attributed to uptake via endogenous Na-K-ATPase and is consistent with
the studies of endogenous Na-K-ATPase in this cell line. As a result,
all 86Rb studies were performed in the presence of 10 µM
ouabain.
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The results of these 86Rb uptake studies demonstrate that
86Rb uptake in pHKc/pHKc
transfected cells was
66-fold greater than that in untransfected cells (118.8 ± 4.0 vs. 1.8 ± 0.3 nmol · mg
protein-1 · 10 min-1). 86Rb uptake in
pHKc
/pNaK
1 transfected cells was also substantially greater
(52-fold) than that in untransfected cells (Fig. 7). Similar to the
observations of H-K-ATPase in the doubly transfected cell lines, uptake
in pHKc
/pHKc
was 26% greater than that in the pHKc
/pNaK
1 cell line. One hundred micromolar vanadate markedly inhibited 86Rb uptake in both doubly transfected cell lines
by ~90%, indicating that 86Rb uptake is mediated by an
ATPase. In contrast, 1 mM ouabain did not significantly alter
86Rb uptake, establishing that 86Rb uptake in
both doubly transfected cell lines is ouabain insensitive. These data
establish that HKc
, when coexpressed with a
-subunit, expresses a
ouabain-insensitive H-K-ATPase function.
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DISCUSSION |
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In addition to the kidney, the mammalian large intestine contributes to
the regulation of overall K balance via both absorptive and secretory
processes (11, 27, 28). Active K absorption that is energized by one
(or more) apical membrane H-K-ATPases has been identified in the distal
colon of rat, guinea pig, and rabbit and has been the focus of several
investigations during the past decade (1, 9, 26, 29). HKc has been
cloned and is a member of the gene family of P-type ATPases (8). HKc
mRNA and protein are present exclusively in surface (and ~20% of the
upper crypt) epithelial cells, and its enzymatic activity is localized
to the apical membrane (15, 18, 24). Considerable controversy exists
regarding whether this HKc
cDNA encodes a ouabain-sensitive or
ouabain-insensitive H-K-ATPase and whether a colon-specific
-subunit
is required for maximal function of the colonic H-K-ATPase (5, 7, 18).
Studies of the coexpression of HKc with either HKg
or NaK
1
cRNAs in Xenopus oocytes yielded evidence of ouabain-sensitive 86Rb uptake that was consistent with H-K-ATPase function
(5, 7). In contrast, expression of HKc
cDNA in Sf9 cells without any
exogenous
-subunit resulted in ouabain-insensitive H-K-ATPase activity (18). It should be noted that in these present studies with
HEK-293 cells, H-K-ATPase activity in cells stably transfected with
pHKc
alone (Fig. 4) was 21% of its activity in cells stably transfected with pHKc
/pHKc
. It is not known whether the
H-K-ATPase activity in the absence of a transfected
-subunit
reflects the presence of an endogenous
-subunit in both Sf9 and
HEK-293 cells or the ability of HKc
protein to manifest partial
H-K-ATPase activity in the absence of any
-subunit.
Recent studies have established the distribution of H-K-ATPase activity
in apical membranes of both surface and crypt cells (22). Although
H-K-ATPase activity in surface cell apical membranes was both ouabain
sensitive and ouabain insensitive, H-K-ATPase activity in apical
membranes of crypt cells was exclusively ouabain sensitive. As
previously noted, HKc mRNA and protein are primarily present in
surface and not in crypt cells (15, 18, 24). Therefore, if HKc
cDNA
encodes a ouabain-sensitive H-K-ATPase, it would be necessary to
postulate the presence of three distinct H-K-ATPases in the rat distal
colon, two that are ouabain sensitive and one that is ouabain
insensitive. Alternatively, however, the presence of only
two H-K-ATPases would be required if HKc
cDNA encoded the
ouabain-insensitive H-K-ATPase. At the present time, there is evidence
for at least two H-K-ATPase isoforms in the rat distal colon.
A -subunit, HKc
, was recently cloned and identified from rat
colon (23). A closely related isoform has been designated by others as
NaK
3 (19). Although HKc
mRNA is present in high abundance in
testis and lung, several lines of evidence have been presented that
provide the basis for the suggestion that HKc
is the
-subunit for
the colonic H-K-ATPase (23): 1) HKc
protein is present in
both apical and basolateral membranes of rat distal colon; 2)
HKc
protein was coprecipitated with HKc
protein from apical
membranes; 3) HKc
mRNA and its apical membrane protein were
increased in distal colon of K-depleted rats (23). Thus it is likely
that the HKc
is the
-subunit for H-K-ATPase in native tissue. The
present study sought to establish whether HKc
cDNA, when
cotransfected with HKc
cDNA in a mammalian cell line, expressed both
H-K-ATPase activity and 86Rb uptake that were inhibited by
vanadate but not by ouabain.
These present studies were designed to express HKc cDNA in a
mammalian expression system in view of the conflicting observations previously reported in the expression of HKc
cDNA in nonmammalian cells. The initial studies with COS-7 cells did not result in the
expression of H-K-ATPase activity but did provide important information
that was critical in the design of the subsequent experiments with
HEK-293 cells. Such cells had been successfully used for the expression
of other P-type ATPases, e.g., ATP1AL1 (13).
Immunofluorescence studies confirmed the presence of both HKc and
HKc
proteins in plasma membranes (Fig. 3). H-K-ATPase activity was
minimal in the nontransfected HEK-293 cells and those transfected with
only the HKc
cDNA. In contrast, transfection of HKc
cDNA resulted
in H-K-ATPase activity both in the absence or presence of one of the
-subunits. Maximal H-K-ATPase activity was observed in those cells
stably transfected with pHKc
/pHKc
and was 60% greater than
that determined in the cells stably transfected with pHKc
/pNaK
1
(Fig. 4). Because equal amounts of HKc
protein were present in the
crude plasma membranes of pHKc
/pHKc
and pHKc
/pNaK
1
transfected cell lines (Fig. 2A, lanes 5 and
6), the observed differences in H-K-ATPase activities in these
doubly transfected cell lines cannot be attributed to differences in HKc
protein expression. Thus the higher rate of H-K-ATPase activity observed when HKc
cDNA was cotransfected with HKc
than
with NaK
1 is consistent with the recent suggestion
(23) that HKc
is the
-subunit for colonic H-K-ATPase.
HKc protein is not the only
-subunit to combine with HKc
protein and to manifest H-K-ATPase activity. Codina et al. (4) recently
demonstrated that HKc
assembles with NaK
1 protein in the rat
distal colon and kidney medulla. In addition, in the present studies
the coexpression of HKc
with NaK
1 resulted in H-K-ATPase function, indicating that NaK
1 protein could act as a surrogate
-subunit for HKc
.
Our previous observations in Sf9 cells (18) are similar to the results
shown in Fig. 4, in which H-K-ATPase activity was identified in the
cell line stably transfected with HKc cDNA alone. Although the
experiment shown in Fig. 4 revealed evidence of H-K-ATPase activity in
the absence of an obvious
-subunit, the coexpression of HKc
cDNA
with HKc
cDNA resulted in an almost fivefold higher level of
H-K-ATPase expression. The expression of H-K-ATPase in the absence of a
-subunit would be consistent either with an endogenous NaK
1 in
the HEK-293 cells functioning as a surrogate or promiscuous
-subunit
or with the presence of a previously unidentified
-subunit in the
HEK-293 cells.
These present results demonstrate that both the expressed H-K-ATPase
activity and the expressed 86Rb uptake in the cell lines
stably transfected with pHKc/pHKc
or with pHKc
/pNaK
1
are ouabain insensitive (Figs. 6 and 7) and conflict with prior
observations in Xenopus oocytes that HKc
cDNA encodes a
ouabain-sensitive function (5, 7). It should be noted that all prior
demonstrations of the ouabain-sensitive HKc
cDNA expression used
86Rb uptake as the parameter of HKc
protein expression
(5, 7). In contrast, these present studies directly determined both
H-K-ATPase activity and 86Rb uptake as a surrogate marker
of K uptake. An adequate explanation for this difference is not
evident; however, it is possible that in the nonmammalian
Xenopus oocyte expression system, HKc
cDNA activated an
endogenous ouabain-sensitive function.
K-dependent proton secretion has been described in the guinea pig colon
and has been attributed to an H-K-ATPase (26). Recently, a cDNA from
guinea pig distal colon (25), which has significant (88%) sequence
homology at the protein level to rat HKc, was expressed in HEK-293
cells with Torpedo Na-K-ATPase
-subunit cDNA and was associated with
ouabain-sensitive H-K-ATPase activity. The rat and guinea pig colonic
H-K-ATPase
-subunit cDNAs share significant sequence homology;
however, it would be important to know whether the guinea pig
H-K-ATPase
-subunit is present in surface or crypt cells.
Physiological studies in the rat indicate the presence of two distinct
H-K-ATPases, one that is ouabain sensitive and present in crypt cells
and the other that is ouabain insensitive and present in surface cells
(22). The encoded proteins from rat and guinea pig respond differently
to ouabain and, therefore, these two proteins likely represent
different H-K-ATPase isoforms.
Two nongastric H-K-ATPase (colonic H-K-ATPase and ATP1AL1) -subunits
with a
-subunit have recently been shown to manifest Na/K exchange
function (6, 14) in addition to their H/K exchange activity. Neither
study assessed whether the expressed proteins had ATPase activity.
Although this present study identified H-K-ATPase activity in the
stably transfected HEK-293 cell lines, transfection with these
- and
- subunits did not result in an increase of the expression of
Na-K-ATPase activity above that in untransfected cells. It is possible
that these different results reflect the different
-subunits used in
these studies.
In conclusion, this study demonstrated that HKc and HKc
cDNAs,
when expressed in a mammalian cell system, manifest ouabain-insensitive H-K-ATPase functions, both enzymatic ATPase activity and
86Rb uptake.
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ACKNOWLEDGEMENTS |
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We thank Dr. John P. Hayslett and Lawrence J. Macala for allowing
us to use their tissue culture facility. HEK-293 cells were kindly
provided by Dr. Bliss Forbush. NaK1 cDNA and NaK
1 antibody were
kindly provided by Dr. Michael Caplan.
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FOOTNOTES |
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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-18777-24.
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.
Address for reprint requests and other correspondence: H. J. Binder, Section of Digestive Diseases, Dept. of Internal Medicine, Yale Univ. School of Medicine, 333 Cedar St., 89 LMP, New Haven, CT 06520-8019 (E-mail: binder{at}biomed.med.yale.edu).
Received 23 March 1999; accepted in final form 20 September 1999.
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