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J Biol Chem, Vol. 274, Issue 28, 19693-19698, July 9, 1999
From the Recent studies have suggested that the colonic
H+,K+-ATPase (HK Both the distal colon and renal medulla participate importantly in
K+ homeostasis (1). A unique
H+,K+-ATPase
(HK HK Cougnon et al. (14) demonstrated that HK High levels of expression of HK Our results demonstrate that these membranes contain both
Na+-dependent and Na+-independent
ATPase activities. Using an inhibitory antibody specific for
HK Preparation of Plasma Membranes (Method I)
To prepare plasma membranes (18, 19) rat distal colon (1 g of
tissue) was homogenized using a Brinkmann Polytron, Model PT 10/35,
followed by homogenization in a Dounce homogenizer using pestle A (4-5
strokes). The homogenization was performed in 10 ml of buffer A (10 mM Tris-HCl, pH 8.0, 1 mM EDTA-Tris, 1 mM phenylmethylsulfonyl fluoride, 3 mM
benzamidine, and 1 µg/ml soybean trypsin inhibitor) containing 27%
sucrose (w/v). Nuclei were removed by centrifugation at 2000 × g for 4 min at 4 °C, the supernatant was applied to the
top of 45% (w/w) sucrose in buffer A and centrifuged at 200,000 × g for 45 min at 4 °C. The membranes in the interphase
27/45% sucrose were diluted in buffer A and collected by
centrifugation at 25,000 × g, resuspended in buffer A,
and stored in aliquots at Preparation of Apical Membranes (Method II)
Apical membranes from distal colon were prepared as described by
Aronson (21). Distal colon (1 g in 10 ml of buffer B: 300 mM mannitol, 1 mM Tris-HCl, pH 7.2, 1 mM phenylmethylsulfonyl fluoride, 3 mM
benzamidine, and 1 µg/ml soybean trypsin inhibitor) was homogenized
with a Polytron, followed by 4-5 strokes with pestle A of a Dounce
homogenizer. The particulate matter was removed by centrifugation for 2 min at 200 × g. To the supernatant, 1 M
MgSO4 was added to a final concentration of 10 mM MgSO4. The sample was placed on ice for 15 min and was shaken intermittently. The aggregated material was removed
by centrifugation for 12 min at 2500 × g at 4 °C,
and apical membranes from the supernatant were collected by
centrifugation at 27,000 × g for 20 min at 4 °C.
The pellet was resuspended in 5 ml of buffer B containing 10 mM MgSO4, and homogenized with pestle B of a
Dounce homogenizer. After removal of the aggregated material at
3100 × g for 12 min at 4 °C, membranes were
collected by centrifugation at 27,000 × g for 20 min
at 4 °C. The final membranes were resuspended in 10 mM
Tris-HCl, pH 7.2, 1 mM EDTA-Tris, 1 mM
phenylmethylsulfonyl fluoride, 3 mM benzamidine, and 1 µg/ml soybean trypsin inhibitor and stored in aliquots at Preparation of Vesicle Membranes (Method III)
Distal colon was homogenized with a Polytron in 3 volumes of
iced 0.25 M sucrose, 1 mM EDTA, as described
previously in our laboratory (22). The homogenate was filtered through
500-µm pore size mesh nylon and centrifuged at 8,000 × g. The supernatant was retained and centrifuged at
200,000 × g. The pellet was resuspended in 250 mM sucrose, 6 mM histidine, pH 7.0, and
recentrifuged at 200,000 × g for 30 min at 4 °C.
The new pellet was resuspended in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA and stored at ATPase Assays
All ATPase assays were performed for 30 min at 37 °C in a
final volume of 200 µl containing 30 mM Tris-HCl, pH 7.2, 1 mM EDTA-Tris, 0.1 mM EGTA-Tris, 4 mM MgCl2, 3 mM ATP-Tris containing
1-10 × 106 cpm of [ K+-ATPase Activity in the Presence or Absence of
Anti-HK Group A--
Serum (15 µl) containing the
anti-HK Group B--
H2O (10 µl) was mixed with serum (15 µl) containing the anti-HK Other Reagents
The characterization of the antibody against HK Distal Colon Apical Membranes Are Enriched in
HK K+-ATPase Activities in Distal Colon Apical
Membranes--
In heterologous expression systems, the activity of rat
HK
Based on the observation that many transporters of K+,
including the
The activity of Na+-dependent
K+-ATPase in apical membranes was inhibited by high
concentrations of ouabain (IC50 ~ 200-300 µM). Replacement of K+ by
NH4+ did not alter this inhibitory
profile (Fig. 4). Furthermore, Sch-28080
did not inhibit K+-ATPase activity when K+ was
replaced by NH4+ (data not shown).
The studies shown above (Figs. 2-4) demonstrate that apical membranes,
which are enriched in HK
To confirm further that HK Our studies demonstrate that plasma membranes from distal colon,
which are enriched severalfold in HK Precedent exists for Na+ secretion by the
HK It is appreciated generally that Na+,K+-ATPases
are localized to basolateral membranes (30). In the present study, and
from previous studies by our laboratory (10) it has been demonstrated that HK Del Castillo et al. (32) reported that two types of
Na+-independent K+-ATPases are present in
apical membranes from distal colon: one which is sensitive to ouabain
(1 mM), and another which is insensitive to ouabain (1 mM). These K+-ATPases were not present in
basolateral membranes. The effect of Sch-28080 (a standard inhibitor of
the gastric H+,K+-ATPase) was not tested. Lee
et al. (33) reported that in preparations of "apical"
membranes from distal colon, both activities (ouabain-insensitive and
ouabain-sensitive K+-ATPases) were inhibited by an antibody
directed against the amino-terminal of HK In agreement with these findings, both
Na+-dependent and Na+-independent
K+-ATPases were detected in the present study, the
Na+-dependent fraction, which predominated
under the conditions of the assay, was relatively ouabain-sensitive. In
contrast, the less abundant Na+-independent fraction was
ouabain-insensitive. Based on the present study it is reasonable to
conclude that HK An additional finding in the present study was that the
K+-ATPase activity in distal colon membranes also had high
affinity for NH4+ (Figs. 3-5).
Transport of NH4+ by the colonic
H+,K+-ATPase in kidney has been suggested in
preliminary studies previously (35, 36). Our findings are the first to
suggest that substitution of K+ by
NH4+ may occur in distal colon apical
membranes. Nevertheless, the physiological role of
H+/NH4+ exchange by the
colonic H+,K+-ATPase in either kidney or distal
colon has not yet been defined clearly.
*
This work was supported in part by National Institutes of
Health, National Institute of Diabetes, Digestive and Kidney Diseases, Grant DK-30603 (to T. D. D.).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.
¶
To whom correspondence should be addressed: Div. of Renal
Diseases and Hypertension, University of Texas-Houston Medical School, 6431 Fannin St., Rm. 4.148, Houston, TX 77030. Tel.: 713-500-6868; Fax:
713-500-6882; E-mail: tdubose@heart.med.uth.tmc.edu.
The abbreviations used are:
HK
The Colonic H+,K+-ATPase Functions as a
Na+-dependent
K+(NH4+)-ATPase in
Apical Membranes from Rat Distal Colon*
,¶
Division of Renal Diseases and Hypertension
Department of Internal Medicine, University of Texas, Houston
Medical School, Houston, Texas 77030 and the § Department
of Physiology, Texas Tech University Health Sciences Center,
Lubbock, Texas 79430
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2) can
secrete either Na+ or H+ in exchange for
K+. If correct, this view would indicate that the
transporter could function as either a Na+ or a
H+ pump. To investigate this possibility a series of
experiments was performed using apical membranes from rat colon which
were enriched in colonic H+,K+-ATPase protein.
An antibody specific for HK2 was employed to determine
whether HK2 functions under physiological conditions as
a Na+-dependent or Na+-independent
K+-ATPase in this same membrane fraction.
K+-ATPase activity was measured as
[
-32P]ATP hydrolysis. The
Na+-dependent K+-ATPase accounted
for approximately 80% of overall K+-ATPase activity and
was characterized by insensitivity to Sch-28080 but partial sensitivity
to ouabain. The Na+-independent K+-ATPase
activity was insensitive to both Sch-28080 and ouabain. Both types of
K+-ATPase activity substituted
NH4+ for
K+ in a similar manner. Furthermore, our results
demonstrate that when incubated with native distal colon membranes, the
blocking antibody inhibited dramatically
Na+-dependent K+-ATPase activity.
Therefore, these data demonstrate that HK2 can function
in native distal colon apical membranes as a
Na+-dependent K+-ATPase.
Elucidation of the role of the pump as a transporter of Na+
versus H+ or
NH4+ versus
K+ in vivo will require additional studies.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2)1 was
cloned by Crowson and Shull (2) from a rat distal colon library, which
was distinct at the amino acid level from both 1-Na+,K+-ATPase (73%
similarity) and from gastric H+,K+-ATPase (72%
similarity). It has been demonstrated, using the Xenopus
laevis oocyte as an expression system, that HK2 can
internalize Rb+ (K+) in exchange for
H+ when coexpressed with any known
X+,K+-ATPase 
-subunit (3-5). Furthermore,
HK2 was insensitive to Sch-28080, a specific inhibitor
of the gastric H+,K+-ATPase, but
partially sensitive to ouabain (IC50 ~ 400-600
µM) (3, 4), a specific inhibitor of the
Na+,K+-ATPase.
2 mRNA and protein are expressed in low abundance
in the renal medulla (2, 6, 7). However, HK2 mRNA
and protein abundance in the kidney are dramatically augmented by
chronic dietary K+-depletion (6, 8-10). This finding
indicates that HK2 may play a major role in renal
K+ conservation. Furthermore, the site of this regulatory
response to chronic hypokalemia has been shown to be the renal medulla (10). Based on data obtained using heterologous expression systems it
has been predicted that the increase in K+-reabsorption in
the collecting tubule during chronic hypokalemia would be insensitive
to Sch-28080 in vivo. Nevertheless, several laboratories
which have evaluated HCO3
reabsorption
during chronic hypokalemia have defined
H+,K+-ATPase function by its sensitivity to
Sch-28080. For example, Wall et al. (11) and Nakamura
et al. (12) have demonstrated that the increase in
bicarbonate absorption
(JtCO2) observed in the
medullary collecting duct during chronic hypokalemia is inhibited by
Sch-28080 in low concentration (10 µM). Sensitivity to
low concentrations of Sch-28080 is characteristic of the gastric H+,K+-ATPase (13). Nevertheless, it has been
suggested that in the inner medullary collecting tubule
HK2 may become sensitive to low concentrations of
Sch-28080 during chronic hypokalemia (12).
2 can
also function to secrete Na+ in exchange for
K+. These findings were supported by studies from Grishin
and Caplan (15), which demonstrated that HEK-293 cells co-transfected
with human ATP1AL1 (90% similar to HK2) and the rabbit
-subunit of the gastric H+,K+-ATPase, grow in the
presence of ouabain. This observation suggests that in transfected
cells, during inhibition of the native Na+ pump by ouabain,
ATP1AL1 which is relatively insensitive to ouabain (16, 17), functions
as a Na+ pump. A possible physiological role for the rat
HK2 or the human ATP1AL1, as apical Na+
pumps, is difficult to envision. Moreover, that these findings were
obtained using heterologous expression systems, raises concern regarding the possibility that a similar function might not exist in
native membranes.
2 have been reported in
apical membranes from rat distal colon (10) where the protein has also
been identified by immunolocalization (9). Taking advantage of this
observation, we prepared apical membranes from rat distal colon to
determine: (a) if under physiological conditions
HK2 is sensitive to low concentrations of Sch-28080, and
(b) if HK2 function, here defined as
K+-ATPase enzymatic activity, is
Na+-dependent or Na+- independent.
2, we demonstrate that this pump is responsible for
Na+-dependent activity. This finding indicates
that the colonic H+,K+-ATPase, which has been
shown to function as a proton pump, may also function as a sodium pump
under certain physiological conditions. Furthermore, we also
demonstrate that both Na+-dependent and
Na+-independent activities can substitute readily
NH4+ for K+.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C. The final protein concentration
was measured using the method of Lowry et al. (20).
70 °C.
The protein concentration was measured using the method of Lowry
et al. (20).
70 °C. The protein concentration
was measured using the method of Lowry et al. (20) and
immunoblots were performed using a specific antibody against
HK2 (10).
-32P]ATP
(Amersham Pharmacia Biotech, catalog number AA0068), 1 mM N-ethylmaleimide, 10 µg/ml oligomycin, 1 mM
phenylmethylsulfonyl fluoride, 3 mM benzamidine, 1 µg/ml
soybean trypsin inhibitor and, when necessary, ouabain, Sch-28080,
NaCl, and/or KCl were added (see figure legends for the concentrations
of ouabain, Sch-28080, NaCl, and KCl). The reaction was started by
addition of rat distal colon apical membranes (5-15 µg) diluted in
10 mM Tris-HCl, pH 7.2. The reaction was stopped by
addition of activated charcoal (1 ml, 50% slurry) (Fisher Scientific,
catalog number C170-500) in 10 mM
Na+-phosphate, pH 7.5. The samples were vortexed, cooled in
ice, centrifuged, and the supernatant (450 µl) was used to quantify the 32P released during the incubation.
2 Antibody
2 antibody was mixed with the immunizing peptide
(10 µl) (1.5 mM) to inactivate the antibody (19), the
antibody/synthetic peptide mixture was incubated for 1 h at
4 °C. After incubation, distal colon apical membranes (100-200 µg) were added to the mixture and incubated for 1 h at 4 °C
with occasional vortexing. The membranes were diluted with 10 mM Tris-HCl, pH 7.2, to a final concentration of 0.5-1.5
µg of protein/µl and 20 µl of the diluted membranes were used in
the ATPase assays.
2 antibody. After 1 h
incubation at 4 °C, rat distal colon apical membranes (100-200
µg) were added and incubated for 1 additional hour at 4 °C. The
membranes were then diluted with 10 mM Tris-HCl, pH 7.2, to
a final concentration of 0.5-1.5 µg of protein/µl. Finally,
immunizing peptide (10 µl) was added. A graphic representation of
this assay is shown in the top panel of Fig. 5. A volume of 20 µl of diluted membranes was used in the ATPase assays.
2
has been described recently by our laboratory (10, 19). Immunoblots were performed as reported previously by our laboratory (10, 19).
Harlan Sprague-Dawley male rats (150-200 g) were used in all
experiments. Ouabain was purchased from Sigma. Sch-28080 was a gift
from Dr. Kaminski at Schering-Plow Research Institute. The anti-NASE
and anti-LEAVE antibodies were characterized previously (23). The amino
acid alignment between different -subunits was performed with the
"Bestfit" program of the Genetics Computer Group (Madison, WI).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2--
As displayed in Fig.
1, plasma (method I), apical (method II),
and vesicle (method III) membranes (20 µg/each) were applied to a
10% SDS-PAGE, transferred to a nitrocellulose membrane and incubated
with the anti-HK2 antibody (dilution 1:1000). As
expected (10), of the three methods to prepare membranes the apical
membrane method yielded membranes which were most enriched in
HK2 protein.
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Fig. 1.
Distal colon apical membranes are enriched in
HK 2. Membranes prepared
according to Methods I, II, or III (20 µg) were resolved on a 10%
SDS-PAGE, transferred to a nitrocellulose membrane, and incubated with
anti-HK2 (dilution 1:1000). The presence of the protein
was determined using an ECL system.
2 has been reported to be
K+-dependent (IC50 < 1 mM), Sch-28080 insensitive, and only partially sensitive to
ouabain (IC50 ~ 400-600 µM) (3, 4). To
test which of the different K+-ATPases observed in apical
membranes of distal colon best fits this pharmacological profile, we
measured Na+-dependent and
Na+-independent K+-ATPase activity in
preparations of rat distal colon apical membranes in the presence of
KCl (10 mM). The data displayed in Fig.
2 (left panel) demonstrate
that the predominant K+-ATPase in distal colon apical
membranes was Na+-dependent and was abolished
by ouabain (2 mM), but was insensitive to Sch-28080 (100 µM). This same figure also demonstrates that distal colon
apical membranes contain a Na+-independent
K+-ATPase. The Na+-independent
K+-ATPase represents only 20% of the total
Na+-dependent K+-ATPase activity
(at 10 mM KCl), and is insensitive to both ouabain (2 mM) and Sch-28080 (100 µM). Treatment of the
membrane preparations with CHAPS or Triton X-100 did not alter the
response to Sch-28080 or ouabain for either
Na+-dependent or Na+-independent
K+-ATPase fractions. Since Sch-28080 did not inhibit either
K+-ATPase activity in apical membranes from distal colon,
we tested if the Na+-independent K+-ATPase
("H+,K+-ATPase") in apical membranes of rat
stomach which are enriched in HK1 is sensitive to
Sch-28080. The right panel of Fig. 2 demonstrates that
concentrations of Sch-28080 as low as 10 µM inhibit
80-90% of the Na+-independent K+-ATPase
activity in the presence of K+ (5 mM).
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Fig. 2.
Left panel, rat distal colon apical
membranes contain both Na+-dependent and
Na+-independent K+-ATPases. The ATPase assay
was performed as described under "Experimental Procedures."
Right panel, rat stomach apical membrane. In contrast to the
Sch-28080 insensitivity of both K+-ATPases in distal colon,
Sch-28080 has high affinity for rat gastric
H+,K+-ATPase (HK 1), as expected.
The assay was performed as described under "Experimental
Procedures" with the only exception that all Na+ salts
were avoided. 1, 1-subunit of the
Na+,K+-ATPase.
1-Na+,K+-ATPase,
can transport either K+ or
NH4+ with similar affinity (24-26), we
characterized the ion dependence of the K+-ATPases present
in our preparation of distal colon apical membranes. In Fig.
3 (left panel) apical
membranes were incubated in the presence of NaCl (20 mM)
and with increasing concentrations of KCl (closed diamonds)
or NH4Cl (closed squares).
Na+-dependent K+-ATPase activity
increased with increasing concentrations of K+ and reached
saturation at concentrations near 5 mM. Substitution of
K+ by NH4 also induced an increase in
Na+-dependent
NH4+-ATPase in a
concentration-dependent manner. The Vm for K+ and NH4+ are similar.
However, the enzyme had a higher affinity for K+
(IC50 ~ 0.2 mM) than for
NH4+ (IC50 ~ 2 mM). The affinity for Na+ was identical
(IC50 ~ 5 mM) whether K+ or
NH4+ was used in the ATPase assay (Fig.
3, middle panel). Finally we determined if K+
can be replaced by NH4+ in the
Na+-independent K+-ATPase fraction. The results
of a representative experiment are displayed in Fig. 3 (right
panel). The Na+-independent K+-ATPase
activity displayed a low affinity for K+ (activity was not
detectable until [K+] was >2.5 mM,
IC50 > 10 mM, and did not reach saturation
until 40 mM [K+]). Similar results were
obtained when K+ was replaced by
NH4+.
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Fig. 3.
Left panel, the
Na+-dependent K+-ATPase has high
affinity for both K+ and
NH4+. The assay was performed in the
presence of NaCl (20 mM) with increasing concentrations of
KCl or NH4Cl. Center panel, HK 2
has similar affinity for Na+ in the presence of either
K+ or NH4+. The assay was
performed in the presence of KCl (5 mM) or
NH4Cl (5 mM) with increasing concentrations of
NaCl. Right panel, the Na+-independent
K+-ATPase fraction can employ either K+ or
NH4+ but at relatively low affinities.
The assay was performed in the absence of Na+.
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Fig. 4.
Na+-dependent
K+-ATPase in the apical membrane has a low affinity for
ouabain. The effect of ouabain was measured in the presence of KCl
(10 mM) or NH4Cl (10 mM) plus NaCl
(20 mM).
2 protein, display
pharmacological properties which are virtually indistinguishable from
1-Na+,K+-ATPase (27, 28). To
exclude the possibility that we were studying
1-Na+,K+-ATPase activity (which
are contaminating the membranes preparation) rather than the activity
of HK2, we took advantage of two observations made by
our laboratory: (a) our anti-HK2 antibody can
immunoprecipitate the
HK2/1-Na+,K+-ATPase
complex in both the renal medulla and distal colon (19) and,
(b) this same anti-HK2 antibody does not
cross-react or immunoprecipitate
1-Na+-K+-ATPase or any other
X+,K+-ATPase characterized thus far (19). We
asked whether the anti-HK2 antibody could block
Na+-dependent K+-ATPase activity.
To answer this question, the anti-HK2 antibody (15 µl)
was incubated in the presence (group A) or absence of immunizing
peptide (500 µM) (group B) for 1 h at 4 °C (10). Incubation was followed by addition of membranes (100 µg) to each group. The mixture was then incubated for 1 h at 4 °C.
Membranes were diluted to 0.5 µg/µl in the presence of 10 mM Tris-HCl, pH 7.2, and incubated at 37 °C for 30 min
as described under "Experimental Procedures." In group B the
immunizing peptide was added after dilution of the membranes. This
approach was taken to correct for possible interference of the peptide
with the ATPase assay. A schematic representation of this protocol is
displayed in Fig. 5 (top
panel). Fig. 5 (middle panel), demonstrates that
incubation of apical membranes with the anti-HK2
antibody markedly decreased Na+-dependent
K+-ATPase activity (closed bar). A similar
experiment was performed by substituting K+ for
NH4+ (Fig. 5, lower panel).
Similar Na+-dependent
K+(NH4+)-ATPase activity was
observed in the presence of either K+ or
NH4+ (5 mM) under control
conditions. Na+-dependent
NH4+-ATPase activity was blocked by the
anti-HK2 antibody, exactly as observed in the presence
of K+. As a control we repeated the experiment described in
Fig. 5, but with plasma membranes isolated from rat renal cortex. These membranes, in contrast to distal colon membranes, are depleted of
HK2 but are enriched in
1-Na+,K+-ATPase. The result of
such an experiment is displayed in Fig. 6. This finding demonstrates that
Na+,K+-ATPase activity in the renal cortex is
not inhibited by the anti-HK2 antibody. Thus, the
anti-HK2 antibody does not cross-react with a membrane
fraction enriched in
1-Na+,K+-ATPase.
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Fig. 5.
Top panel, schematic representation of
the protocol to determine if the Na+-dependent
K+-ATPase activity in distal colon apical membranes
represented HK 2. For differences in two groups (A and B)
see "Experimental Procedures." In group A (controls) the immunizing
peptide was added prior addition of the anti-HK2
antibody and membranes to block recognition of HK2
protein in the membrane by the antibody. Therefore, only the group B
protocol allowed exposure of the native HK2 to the
anti-HK2 to the anti-HK2 antibody.
Middle panel, the Na+-dependent
K+-ATPase of HK2 was blocked by the
anti-HK2 antibody. Bottom panel, the
Na+-dependent K+-ATPase was blocked
by the anti-HK2 antibody when K+ was
replaced by NH4+.
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Fig. 6.
The
anti-HK 2 antibody does not block
Na+,K+-ATPase activity in plasma membranes from
renal cortex. The assay was performed exactly as described in the
legend to Fig. 5.
2 functions as a
Na+-dependent K+-ATPase in apical
membranes of distal colon origin we performed an additional experiment
described in Fig. 7. In this study
Na+-dependent K+-ATPase activity
was measured in plasma membranes from renal cortex as well as apical
membranes from distal colon. We reasoned that if the HK2
functions in distal colon as a Na+-dependent
K+-ATPase, more 1-subunit from the renal
cortex would be required to reach the same level of
Na+-dependent K+-ATPase activity
present in the distal colon apical membranes. Fig. 7 (left
panel) demonstrates that both membrane fractions contain similar
specific activities (expressed as nanomole of ATP hydrolyzed/hour) at
any given concentration of total protein. However, the absolute level
of 1-Na+,K+-ATPase was much
greater in renal cortex compared with distal colon (right
panel of Fig. 7). This study was performed with two different
antibodies, anti-NASE which recognizes only
1-Na+,K+-ATPase and anti-LEAVE
which should recognize 1-, 2-, and
3-Na+,K+-ATPase (23). The
observation that the immunoblots for both the distal colon and renal
cortex membranes using either the anti-NASE and anti-LEAVE antibodies
were indistinguishable, demonstrates that neither 2- nor
3-Na+,K+-ATPase accounts for the
Na+-independent K+-ATPase activity in distal
colon apical membranes. These findings provide additional evidence that
the distal colon apical membrane fraction is enriched in
HK2 protein and functions as a
Na+-dependent K+-ATPase which is
distinct from 1-Na+,K+-ATPase
activity.
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Fig. 7.
The Na+-dependent
K+-ATPase activity in apical membranes from distal colon
cannot be accounted for by
1-Na+,K+-ATPase. Left
panel, Na+-dependent K+-ATPase
activity was similar in both membranes at any concentration of
membranes used. The assay was performed in the presence of KCl (10 mM) and in the presence or absence of NaCl (50 mM). Right panel, abundance of -subunits of
Na+,K+-ATPases in renal cortex exceeds
abundance in distal colon.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 protein, contain a K+-ATPase activity which is
Na+-dependent, Sch 28080-insensitive, and
partially ouabain-sensitive. To conclude that this activity represents
functionally the -subunit of the colonic
H+,K+-ATPase, is contingent on the specificity
of our anti-HK2 antibody. Previous studies by our
laboratory have demonstrated that the antibody used in the present
study does not cross-react with any of the known
X+,K+-ATPases (10, 19). Nevertheless,
alternative explanations for this activity which were evaluated in the
course of this study include all known members of the
X+,K+-ATPase superfamily. The antibody used in
these studies was raised against a synthetic peptide designed after the
rat colonic H+,K+-ATPase the epitope of which
(amino acids 686-698) is not found on any other known rat
X+,K+-ATPases. Furthermore, there is no
evidence that another H+,K+-ATPase or
Na+,K+-ATPase, except HK2 or
1-Na+,K+-ATPase, exists in
distal colon (Fig. 7). Moreover, in this, and in a previous study (10,
19), we have demonstrated that this antibody does not cross-react or
immunoprecipitate other known X+,K+-ATPases,
including the 1-Na+,K+-ATPase.
In addition, we now demonstrate that our anti-HK2
antibody blocks Na+-dependent
K+-ATPase activity (Fig. 5), that
Na+,K+-ATPase activity in the renal cortex is
not inhibited by the specific blocking antibody (Fig. 6), and finally
that neither 2 nor
3-Na+,K+-ATPase could account
for the activity in the distal colon apical membrane fraction (Fig. 7).
Therefore, we conclude that HK2 can function in distal
colon as a Na+-dependent
K+-ATPase.
2. Cougnon et al. (14) has demonstrated
recently that HK2 can secrete Na+ in
exchange for K+ in X. laevis oocytes. In this
study it was reported that Na+/K+ exchange,
which was sensitive to high concentrations of ouabain, and was totally
insensitive to Sch-28080, was dependent on co-expression with a
-subunit. Moreover, Kone and Higham (29) reported recently that a
splice variant of HK2 (HK2b), which is
truncated by the initial 103 amino acids, supported the growth of
HEK-293 cells in the presence of low concentrations of ouabain. In
addition, Grishin et al. (17), expressed the human ATP1AL1
(90% similar to HK2) in HEK-293 cells, and observed
that the ratio of H+-secretion to K+-uptake was
approximately 1:10. Based on these observations he postulated that
ATP1AL1 did not function solely as a H+/K+
exchanger. This group also reported that ATP1AL1 supported the growth
of HEK-293 cells in the presence of ouabain (15). Since HEK-293 cells
transfected with ATP1AL1 or HK2b grow in the presence of
ouabain (15, 29), it is logical to speculate that both the rat
HK2 and the human ATP1AL1 may function as
Na+ pumps. However, since these results were obtained in
heterologous expression systems, questions could be raised regarding
the relevance of these results to native colonic apical membranes.
2 is enriched in apical membrane fractions from
distal colon. Sangan et al. (8) have also demonstrated that
HK2 protein localizes to the apical membrane of
colonocytes. Moreover, Jaisser et al. (31) have demonstrated
by in situ hybridization that HK2 mRNA
localizes to surface epithelial cells of rat distal colon. Assigning a
physiological role for a Na+-dependent
K+-ATPase located in the apical membrane of colonocytes is
difficult, however.
2. Recently,
Rajendran et al. (34) reported that
Na+-independent, K+-dependent
pHi recovery by rat colonocytes was ouabain-insensitive (up to
1 mM). Based on this observation, it was concluded that HK2 functions in colonocytes as a
Na+-independent, ouabain-insensitive
K+-ATPase.
2 may function in native apical
membranes not only as a proton pump, but as a Na+ pump.
Nevertheless, the physiological conditions which might serve to
regulate Na+/K+ as opposed to
H+/K+ exchange have not been defined.
Therefore, future studies will be needed to define the relative
contributions of these pumps in the distal colon as well as factors
which regulate their abundance and function in physiologic and
pathophysiologic conditions.
FOOTNOTES
ABBREVIATIONS
1, -subunit of the gastric H+,K+-ATPase;
HK2, -subunit of the colonic
H+,K+-ATPase;
1, -subunit of
the Na+,K+-ATPase;
PAGE, polyacrylamide gel
electrophoresis;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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