Departments of Internal Medicine and Pathology, Yale University, New Haven, Connecticut 06520-8019
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ABSTRACT |
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P-type ATPases
require both - and
-subunits for functional
activity. Although an
-subunit for colonic apical membrane
H-K-ATPase (HKc
) has been identified and studied, its
-subunit
has not been identified. We cloned putative
-subunit rat colonic
H-K-ATPase (HKc
) cDNA that encodes a 279-amino-acid protein with a
single transmembrane domain and sequence homology to other rat
-subunits. Northern blot analysis demonstrates that this HKc
is
expressed in several rat tissues, including distal and proximal colon,
and is highly expressed in testis and lung. HKc
mRNA abundance is upregulated threefold compared with normal in distal colon but not
proximal colon, testis, or lung of K-depleted rats. In contrast, Na-K-ATPase
1 mRNA abundance is
unaltered in distal colon of K-depleted rats. Na depletion, which also
stimulates active K absorption in distal colon, does not increase
HKc
mRNA abundance. Western blot analyses using a polyclonal
antibody raised to a glutathione
S-transferase-HKc
fusion protein
established expression of a 45-kDa HKc
protein in both apical and
basolateral membranes of rat distal colon, but K depletion increased
HKc
protein expression only in apical membranes. Physical
association between HKc
and HKc
proteins was demonstrated by
Western blot analysis performed with HKc
antibody on
immunoprecipitate of apical membranes of rat distal colon and HKc
antibody. Tissue-specific upregulation of this
-subunit mRNA in
response to K depletion, localization of its protein, its upregulation
by K depletion in apical membranes of distal colon, and its physical
association with HKc
protein provide compelling evidence that HKc
is the putative
-subunit of colonic H-K-ATPase.
active potassium absorption; rat distal colon; hydrogen-potassium-adenosinetriphosphatase -subunit
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INTRODUCTION |
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ACTIVE K ABSORPTION AND secretion are important
transport processes of mammalian large intestine (2). Active K
absorption, a unique function of the distal colon, is energized and
regulated by H-K-ATPase, an apical membrane P-type ATPase (8, 11). This
colonic H-K-ATPase is a member of a gene family of related P-type
ATPases that include Na-K-ATPase and gastric H-K-ATPase (7, 14, 35,
36). ATPases in this gene family usually are heterodimers that consist
of - and
-subunits. The
-subunit contains the catalytic
function of these ATPases, whereas the specific function of the heavily
glycosylated
-subunit is not completely known. Recent studies
indicate that important functional properties of
-subunits include
an essential role in the stabilization, maturation, and enzymatic
activity of both Na-K-ATPase and H-K-ATPase (4).
Colonic K transport is modified by several factors, including changes
in dietary K and aldosterone (11, 12). Increases in dietary K induce
active K secretion, whereas dietary K depletion enhances active K
absorption (12). Aldosterone, either as a result of dietary Na
depletion or following its subcutaneous administration, markedly
stimulates active K absorption in the rat distal colon (11, 37). The
recent cloning of a cDNA that encodes the -subunit of the rat distal
colon H-K-ATPase (HKc
) (7, 14) led to studies that have assessed the
regulation by which dietary Na and dietary K depletion modify active K
absorption and colonic H-K-ATPase (15, 33). Dietary Na depletion
increases HKc
message and protein abundance and H-K-ATPase activity
in the rat distal colon. In contrast, dietary K depletion did not
increase the abundance of HKc
message or protein in the distal colon
(15, 33).
One possible explanation to account for the absence of an upregulation
of HKc message by dietary K depletion is that the effect of dietary
K depletion on active K absorption is mediated by the
-subunit of
the colonic H-K-ATPase, as
-subunits are required for the catalytic
activity of P-type ATPases. Although a specific
-subunit for the
colonic H-K-ATPase has not been isolated, conflicting observations
exist as to whether a colon-specific
-subunit is required for
maximal colonic H-K-ATPase activity (5, 6, 20). Thus, although Lee et
al. (20) recently expressed HKc
in Sf9 cells as ouabain-insensitive
H-K-ATPase activity in the apparent absence of any
-subunit, other
studies in Xenopus oocytes have
demonstrated that the expression of HKc
required noncolonic
-subunits (5, 6). This requirement for such
-subunits may either
represent a promiscuous expression by a related
-subunit, indicating
that the colonic
-subunit is closely related, or indicate that
colonic H-K-ATPase activity requires a
-subunit that is not colon specific.
To identify a -subunit for colonic H-K-ATPase, a series of
low-stringency Northern blot analyses with gastric H-K-ATPase
-subunit (HKg
) and Na-K-ATPase
1- and
2-subunit
(NaK
1 and NaK
2) cDNAs were performed.
These analyses demonstrated either no hybridization (HKg
,
NaK
2) or only a single band
(NaK
1) with mRNA from rat
distal colon. A novel
-subunit cDNA that had been recently cloned
from a rat astrocytoma cell line (38) and that had hybridized with
guinea pig colon mRNA (Y. Suzuki, personal communication) was also used
as a probe in additional Northern blot analyses using mRNA from rat
distal colon. Because this cDNA hybridized with mRNA from rat distal
colon at the size of 1.9 kb, we initiated experiments to clone a rat
colon-derived
-subunit using the rat astrocytoma
-subunit cDNA as
a probe.
The present study demonstrates the following.
1) A full-length -subunit cDNA
(HKc
) isolated from a rat colon cDNA library is identical to a cDNA
isolated from rat astrocytoma cells (38). In studies using this cDNA,
dietary K depletion selectively increases the abundance of HKc
message in the rat distal colon. 2)
A polyclonal antibody to HKc
protein identifies a protein in apical
and basolateral membranes of rat distal colon.
3) This HKc
protein expression is
selectively increased in K depletion in apical but not in basolateral membranes. 4) Coimmunoprecipitation
experiments reveal a physical interaction between HKc
and HKc
proteins. As a result, these observations suggest that this cDNA
encodes the colonic H-K-ATPase
-subunit.
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METHODS |
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Male Sprague-Dawley rats (200-250 g body wt; Charles River Laboratories, Wilmington, MA) were divided into three experimental groups: the control group was fed normal rat food that contained 4.4 g Na/kg and 9.5 g K/kg. The Na-depleted group was given a Na-free diet for 1 wk. The K-depleted group was given a K-free diet (0.6 mg K/kg) for 3 wk. All animals were allowed free access to water.
At the end of the experimental diet periods, the animals were killed and proximal colon, distal colon, ileum, jejunum, stomach, kidney, brain, lung, testis, liver, spleen, and heart from normal rats; proximal and distal colon, testis, and lung from K-depleted rats; and proximal and distal colon from Na-depleted rats were immediately removed and washed with diethyl pyrocarbonate-treated sterilized saline. Colonocytes from proximal and distal colon were obtained, as previously described (33), and all tissues were then homogenized using a Polytron homogenizer in 4 M guanidine isothiocyanate buffer for 60 s and centrifuged at 3,000 rpm for 10 min to remove any unbroken cells. Total RNA was prepared from the supernatant by ultracentrifugation through CsCl (32). Poly(A)+ mRNA was isolated by passing total RNA through oligo(dT) cellulose columns according to the method described by Sambrook et al. (32). Total RNA and mRNA were quantitated by absorbance at 260 nm in an ultraviolet-visible double-beam spectrophotometer (Shimadzu).
Northern blot analysis.
Northern blot analyses were performed using
poly(A)+ mRNA, as previously
described (33). A 32P-labeled
full-length HKc probe [1 × 106
counts · min
1 · ml
1
(cpm/ml)] was added to the membrane for hybridization at 42°C in a Hybaid oven for 18 h. Blots were washed for 15 min in 0.1× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) and 0.1% SDS at 65°C and were exposed to Hyperfilm (Amersham, La
Jolla, CA) using a Bioplus intensifying screen at 70°C.
cDNA screening and sequencing.
A rat colon cDNA library with randomly primed cDNA inserts >1.0 kb in
length was cloned in pcDNAI (Invitrogen, San Diego, CA) and was used in
the cloning of the -subunit; 300,000 colonies were plated onto 20 Luria-Bertani [LB; containing 50 µg/ml ampicillin and 7 µg/ml
tetracycline (amp/tet)] plates 150 mm in diameter. A microwave
colony-screening protocol from Invitrogen was strictly followed.
Briefly, colonies were transferred to nylon membrane (UV Duralon
membrane, Stratagene, La Jolla, CA) after nylon membrane had been
placed on top of the colonies in the plate. Membrane filters were
lifted carefully and were laid on a fresh LB (amp/tet) agar plate by
facing the colony side up; the plates were incubated with filters at
37°C for 1 h to allow colonies to grow. To regrow the colonies on
the agar plates that were lifted, plates were incubated at 37°C for
about 6 h and then stored at 4°C. The filters were carefully
removed from the agar plate and placed colony side up on a Whatman 3 filter paper prewetted in lysis buffer (2× SSC-5% SDS, pH 7.0).
The filter paper and the membrane with colonies were placed in a
turntable microwave oven and heated at high power for 6 min. The
filters were then placed in prehybridization solution containing
2× SSC (pH 7.0), 1% SDS, and 0.5% nonfat dry milk at 65°C
for 1 h. The filters were removed from the prehybridization solution;
any cellular debris was removed. The filters were then hybridized in
buffer containing 6× SSC, 1% SDS, 0.5% nonfat dry milk, 100 µg denatured salmon sperm DNA, and 1 × 106 cpm/ml
32P-labeled full-length
-subunit cDNA probe from a rat astrocytoma cell line (38) labeled by
random primer method (32) at 65°C for at least 16 h. The filters
were then washed at room temperature for 10 min by gentle shaking in a
buffer containing 2× SSC (pH 7.0) and 1% SDS, followed by
1× SSC (pH 7.0) and 1% SDS; the filters were transferred to
prewarmed buffer (45°C) containing 0.1× SSC (pH 7.0) and 1%
SDS and washed by gentle shaking at 45°C for 15 min. The filters
were air dried, covered with Saran wrap, and exposed to Hyperfilm
(Amersham) using a Bioplus intensifying screen for 18 h. After
development of the film, positive colonies were identified and screened
two more times to obtain positive clones. Plasmids were prepared
according to the method of Morelle (27) using 2 ml of "terrific
broth" (32), and the plasmid DNA containing inserts was sequenced in
both strands using an automated fluorescence sequencing machine at the
Yale Sequencing Facility.
Antibody production.
The cDNA sequence (165 bp) corresponding to the amino acid sequence
between Pro-87 and Ser-142 of HKc was amplified using the
full-length colon-derived HKc
as a template with a sense primer
(5'-GGGGGGATCCCACCGACTGCCTTGGATTATACATA-3')
to which a BamH I site was appended at
the 5' end and an antisense primer (5'-GGGGGGAATTCACTATAGTCTGGACCCTCCTG-3')
to which an EcoR I site was appended
at the 5' end. The expected size 165-bp fragment was gel
purified, digested with BamH I and
EcoR I, and ligated using T4 DNA
ligase into PGEX-KG vector that had previously been digested with
BamH I and
EcoR I and dephosphorylated with calf intestine phosphatase. The ligated DNA was transformed into XL-1 blue
Escherichia
coli cells and spread onto
LB-ampicillin plates. Plasmid DNAs were prepared from recombinant
colonies, digested with BamH I and
EcoR I, and electrophoresed onto 1.5%
agarose gels. Positive clones containing the 165-nucleotide HKc
fragment were selected and sequenced. Clones with correct reading frame and with correct cDNA sequence were selected to prepare glutathione S-transferase (GST)-HKc
fusion
protein. The recombinant colonies were grown in LB-ampicillin medium to
an absorbance of 0.8 at 600 nm; the GST-HKc
fusion proteins were
then overexpressed after induction with 1.0 mM isopropyl
-D-thiogalactopyranoside at
37°C for 3 h. After induction, the cells were harvested, washed
with 50 mM Tris · HCl (pH 7.4) containing 10 mM EDTA,
and resuspended in the lysis buffer containing 50 mM
Tris · HCl (pH 7.4), 100 mM NaCl, 5 mM
dithiothreitol, 2 mM EDTA, 2 mM EGTA, and 1 mg/ml lysozyme, mixed 45 min at 4°C, frozen at 80°C, and thawed. Then 1% Triton X-100,
10 mM MgCl2, and 0.1 mg/ml DNase I
were added and incubated at 4°C for an additional 30 min. After
centrifugation, the GST-HKc
fusion protein was purified from the
supernatant by passage through a glutathione-agarose column; after the
column was washed separately with 1× PBS and 1× PBS
containing 1% Triton X-100 and with 50 mM Tris · HCl
(pH 8.0) containing 1 mM EDTA, the GST-HKc
fusion protein was eluted
from the column using 10 mM reduced glutathione (pH 8.0) by incubating
at 4°C with gentle nutation. The eluted GST-HKc
fusion protein
was dialyzed against 1× PBS at 4°C and concentrated using
Centriprep 10. At this stage, the purified GST-HKc
fusion proteins
were run on SDS-PAGE gels and analyzed for homogeneity. The homogeneous
preparation of GST-HKc
fusion proteins was used to inject rabbits to
raise polyclonal antibodies.
Specificity of HKc antibody.
The specificity of the HKc
antibody was established by the following
experiments. 1) In an
immunodepletion experiment, GST-HKc
fusion protein and HKc
antibody were mixed and incubated at room temperature. The
antigen-antibody complex mixture was then used as an antibody source
for the Western blot analysis, which contained apical membranes,
basolateral membranes, or the immunoprecipitate of apical membranes by
HKc
antibody. 2) HKc
cDNA and
NaK
1 cDNA were subcloned into
pcDNA 3.1 (+), which was transfected into COS-7 cells (13). The
expressed HKc
and NaK
1
proteins were used for Western blot analysis with HKc
antibody.
3) Highly purified Na-K-ATPase from
rabbit kidney (18) was run on SDS-PAGE gels and transferred to
nitrocellulose membranes, and Western blot analysis was performed using
HKc
or NaK
1 antibodies.
Isolation of apical and basolateral membranes. Apical and basolateral membranes were isolated by methods previously described in detail (29, 30).
Western blot analysis.
Western blot analyses were performed with HKc, HKc
, and
NaK
1 antibodies using
previously described methods (20, 33). Apical or basolateral membrane
proteins (50 µg) were electrophoresed on SDS-PAGE gels and
transferred to nitrocellulose membranes. Western blot analysis was then
performed with dilution of 1:1,000 HKc
antibody in Tris-buffered
saline-Tween containing 5% nonfat dry milk; anti-rabbit IgG
horseradish peroxidase conjugate (1:5,000 dilution) was used as the
secondary antibody. HKc
antibody-specific protein bands were
visualized by the enhanced chemiluminescence procedure.
Coimmunoprecipitation.
One hundred micrograms of apical membrane proteins of rat distal colon
were resuspended in 1 ml of immunoprecipitation buffer containing 10 mM
Tris · HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM
EGTA, 0.2 mM phenylmethylsulfonyl fluoride, and 1% Triton X-100. One
microliter of HKc antibody was added and incubated at room
temperature for 2 h in a nutator. To the above mixture, 50 µl of
protein A-Sepharose (50% suspension) were added to bind antigen-antibody complex and incubated at room temperature for 1 h in a
nutator. After a 1-min centrifugation, the pellet was washed three
times for 5 min each in the above immunoprecipitation buffer containing
5% nonfat dry milk. The pellet was then washed three times with
immunoprecipitation buffer. Finally, the pellet was resuspended in
2× SDS sample buffer containing 2-mercaptoethanol, boiled for 5 min, and centrifuged briefly, and the supernatant was loaded onto
SDS-PAGE gels. After electrophoresis, proteins were transferred onto a
nitrocellulose membrane and Western blot analysis was performed with
HKc
antibody diluted to 1:1,000.
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RESULTS |
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Isolation and characterization of HKc cDNA clones.
We screened a rat colon cDNA library using a full-length novel
-subunit cDNA isolated from a rat astrocytoma cell line (38) as a
probe and obtained positive clones that were sequenced in both
directions. The full-length cDNA of HKc
consists of 1,728 nucleotides with an open reading frame encoding 279 amino acids, an
initiation codon ATG, an in-frame termination codon (TAG), a
78-nucleotide 5' untranslated region, and an 810-nucleotide 3' untranslated sequence. The sequence of this cDNA is identical to that previously cloned by Watanabe et al. (38) from a rat astrocytoma cell line.
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Expression of HKc subunit mRNA in rat tissues.
Expression of HKc
mRNA was analyzed in several tissues by Northern
blot analysis (Fig. 2). HKc
cDNA probe
hybridized with a transcript of ~1.9 kb in distal colon, proximal
colon, ileum, jejunum, stomach, liver, lung, kidney, brain, testes,
spleen, and heart. It should be noted that HKc
cDNA that was cloned
from colon is 1.73 kb. HKc
message was highly expressed in testis and lung; the message was slightly smaller in size in testis than those
identified in other tissues.
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Regulation of HKc expression.
The identification of a colon-derived putative
-subunit isoform
permitted studies to evaluate the role of HKc
in the transport function of the rat distal colon. Because HKc
is expressed in rat
distal colon, it could be associated with the regulation of K transport
and/or Na transport, which are closely linked to colonic H-K-ATPase and Na-K-ATPase, respectively (2). Expression of HKc
was analyzed by Northern blot with mRNA isolated from
distal colon of normal, dietary Na-depleted, and dietary K-depleted
rats (Fig. 3). Na depletion stimulates Na
and K absorption and K secretion (2, 37), whereas K depletion enhances
only K absorption in distal colon (12). Figure
3A demonstrates that HKc
mRNA
expression is increased in distal colon of K-depleted rats compared
with normal rats. HKc
message expression was normalized to
glyceraldehyde-3-phosphate dehydrogenase expression and quantitated by
densitometric analysis. K depletion resulted in a 3.5-fold increase in
HKc
message expression compared with controls (Fig.
3B). In contrast, HKc
mRNA
abundance in Na-depleted rats was not significantly altered (Fig. 3),
although both dietary K depletion and Na depletion induce comparable
increases in active K absorption in the rat distal colon (11, 12). In parallel studies with mRNA isolated from proximal colon, an organ in
which active K absorption is not present (11), dietary K depletion did
not increase HKc
mRNA abundance (Fig.
4).
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Expression of HKc in apical and basolateral
membranes.
The results of the Northern blot analyses (Fig. 3) are consistent with
the thesis that HKc
is the
-subunit required for colonic
H-K-ATPase function. HKc
has significant (80%) homology to a
recently identified human
-subunit that was designated
NaK
3 (18). This assignment of
NaK
3 was based on chromosomal
localization and sequence relatedness. As a consequence, additional
studies were performed with HKc
to establish whether HKc
is
present in apical and/or basolateral membranes and whether
HKc
is associated with HKc
.
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Identification of physical interaction of HKc and
HKc
.
Experiments were designed to establish whether HKc
protein was
associated with HKc
protein in the apical membrane of distal colon.
Therefore, apical membrane proteins from rat distal colon were
immunoprecipitated with HKc
antibody (see
Coimmunoprecipitation). The
resulting antigen-antibody complex was analyzed by Western blot using
HKc
or NaK
1 antibodies.
Figure 8B
(lane
3) demonstrates the presence of
HKc
protein in the immunoprecipitate, demonstrating the physical
interaction of HKc
and HKc
proteins. In contrast, NaK
1 was not identified in the
immunoprecipitate (Fig. 8C,
lane 3). The specificity of the bands of
the immunoprecipitate was confirmed by performing a Western blot
analysis of the protein blot in Fig.
8C using preimmune serum following
stripping. The preimmune serum identified only the immunoglobulin, as
shown in Fig. 8D,
lane
3. In addition, the immunodepletion
experiments (Fig. 7A) established
the specificity of HKc
antibody to HKc
protein. These
observations are consistent with HKc
functioning as the
-subunit
for the apical membrane localized
-subunit of colonic H-K-ATPase.
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DISCUSSION |
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Active K absorption and an apical membrane H-K-ATPase have been studied
extensively in the rat large intestine. Active K absorption is
restricted to the distal colon and is upregulated both by dietary K
depletion and by aldosterone and Na depletion (11, 12). It is generally
accepted that active K absorption is energized by apical membrane
H-K-ATPase, the -subunit of which (HKc
) was recently cloned (7,
14) and has been studied extensively (5, 6, 15, 20, 33). HKc
message
is restricted to surface (and the upper 20% of the crypt) cells of the
distal colon, whereas its protein is localized to the apical membrane
of surface cells of the rat distal colon (14, 20). Although several
-subunit isoforms of both Na-K-ATPase and noncolonic H-K-ATPase have
been isolated, a colonic H-K-ATPase
-subunit had not previously been identified.
Recent studies have established that dietary Na depletion upregulates
HKc message and protein abundance and apical membrane H-K-ATPase
activity in the rat distal colon but does not alter HKc
message and
protein expression in the kidney (15, 33). In contrast to the effect of
Na depletion on HKc
expression in the colon, dietary K depletion did
not affect HKc
message and protein expression in the rat distal
colon (33). Of potential importance, HKc
protein expression was
enhanced in the principal cell in the kidney of dietary K-depleted rats
compared with control rats. Thus the mechanism by which dietary K
depletion increases active K absorption in the rat distal colon is not
known. Lescale-Matys et al. (21) demonstrated in
LLC-PK1 cells that a decrease in extracellular K concentration was associated with an increase in
NaK
1 message and protein but
not in NaK
1 message and
protein. Their study concluded that regulation of Na-K-ATPase activity by K concentration was pretranslational and that
-subunit synthesis was rate limiting. These observations with
LLC-PK1 cells parallel the present
results with rat distal colon. Dietary K depletion enhances active K
absorption but is associated with an increase in HKc
(Fig. 3) but
not HKc
mRNA abundance (33). As a result, we speculate that the
mechanism of regulation of H-K-ATPase function by dietary K
depletion is mediated via HKc
and not via HKc
.
The data presented in Fig. 3 are consistent with the role of HKc
mRNA in the regulation of H-K-ATPase by dietary K depletion. These
Northern blot analyses using the newly cloned colon-derived
-subunit
establish that HKc
mRNA abundance was enhanced in dietary K-depleted
rats. Specificity of this observation is provided by four important
experiments. First, dietary K depletion did not increase the mRNA
abundance of the NaK
1 subunit
(Fig. 6), indicating that dietary K depletion does not result in a
nonspecific increase in message abundance of all
-subunits. Second,
HKc
mRNA abundance was not increased in Na-depleted rats (Fig. 3),
indicating that the increase in HKc
message was specific and was not
merely secondary to an increase in active K absorption. Third, although
HKc
message is present in the proximal colon (Fig. 4), a tissue in
which active K absorption is not present (11), HKc
mRNA abundance
was not increased in dietary K-depleted animals in proximal colon.
Fourth, expression of HKc
mRNA abundance was not altered in lung and testis of K-depleted rats compared with normal rats (Fig. 5). Thus the
effect of K depletion on HKc
mRNA is tissue specific, as HKc
mRNA
is upregulated in the distal but not in the proximal colon, testis, or
lung of K-depleted rats. These observations provide compelling evidence
that HKc
is closely linked to the stimulation of active K absorption
by dietary K depletion in the rat distal colon.
Controversy exists regarding the role of a -subunit in the
functional expression of HKc
. Although HKc
was expressed as ouabain-insensitive H-K-ATPase in Sf9 cells without the apparent presence of a
-subunit (20), the expression of HKc
in oocytes requires the coinjection of cRNAs of a
Bufo
marinus urinary bladder
-subunit
(6) or of HKg
or NaK
1 (5).
It is of interest that the amphibian
-subunit (17) that induced
86Rb uptake in oocytes when
coinjected with HKc
(6) has significant homology (52.3%) to HKc
.
The demonstration that dietary K depletion resulted in an increase in
HKc
mRNA abundance but not an enhancement of HKc
mRNA abundance
strongly suggests that the regulation of active K absorption by dietary
K depletion is mediated by this
-subunit and not by HKc
. Because
Na depletion stimulates HKc
but not HKc
message abundance in the
rat distal colon (Refs. 15, 33 and Fig. 3), differential regulation of
HKc
and HKc
subunits plays a significant role in the stimulation
of active K absorption in rat distal colon by K depletion and Na
depletion. In addition, because dietary K depletion increased the
abundance of HKc
message but not that of
NaK
1, these observations
suggest that HKc
may be the specific
-subunit required for
optimal H-K-ATPase function in the distal colon.
The close identity between HKc and the recently reported human
putative NaK
3 (2, 22) requires comment. The latter cDNA sequence was identified from the human-expressed sequence tag data bank
and was designated NaK
3 based solely on chromosomal localization and sequence relatedness, but without demonstration of
membrane localization or physical association with an
-subunit. In
addition, this human putative NaK
3 has an amino acid
sequence with a 55% identity to a recently cloned
-subunit from
amphibian bladder (17), which, when coexpressed in Xenopus
oocytes with an amphibian bladder H-K-ATPase
-subunit, stimulated
H/K and not Na/K function (16). Because HKc
protein has 52%
identity to the amphibian bladder
-subunit, HKc
may be the rat
homologue of this amphibian bladder
-subunit. Thus it is possible
that the human putative NaK
3 may also manifest
H-K-ATPase function.
The identification of HKc protein in basolateral membranes (see Fig.
8B,
lane
2) raised the possibility that
HKc
might also function with
-subunits of Na-K-ATPase and that
therefore the upregulation of HKc
mRNA by dietary K depletion (Fig.
3) was causally related to an increase in Na-K-ATPase activity by
dietary K depletion. Such a possibility would be consistent with the
previous observations that dietary K depletion and incubation of
LLC-PK1 cells in a low-K medium
are associated with increases in
NaK
1 subunit and/or
Na-K-ATPase activity (21). Therefore, additional experiments were
performed to determine whether dietary K depletion was associated with
an increase in Na-K-ATPase activity in rat distal colon. These studies
demonstrated that Na-K-ATPase activity in basolateral membranes
isolated from rat distal colon was not altered by dietary K depletion
(unpublished observations). Thus the observed increase in HKc
mRNA
abundance in dietary K depletion cannot be responsible for an increase
in Na-K-ATPase. Therefore, HKc
is uniquely regulated by dietary K
depletion in the distal colon and likely is the
-subunit required
for H-K-ATPase in the rat distal colon.
H-K-ATPase is localized to the apical membrane in the distal colon (8),
in contrast to the localization of Na-K-ATPase to the basolateral
membrane. The Western blot studies presented in Fig. 8 present three
important observations that indicate HKc is the
-subunit for the
colonic H-K-ATPase. First, HKc
protein was expressed in apical
membrane (Fig. 8B,
lane
1), the site at which HKc
protein
is selectively expressed (see Fig. 8A,
lane 1). Second, although HKc
protein
was identified in both apical and basolateral membranes (Fig.
8B,
lanes
1 and
2), K depletion resulted in an
increase in HKc
protein expression only in apical membranes (Fig.
9). The selective increase in HKc
protein in the apical membrane
requires comment. It is possible that the protein recognized by the
HKc
antibody in the basolateral membrane is not HKc
protein but a
closely related
-subunit that was recognized by the HKc
antibody
but not regulated by K depletion. The mechanism for the 30% decrease
of HKc
protein in basolateral membrane of K-depleted rats is not
known. Third, coimmunoprecipitation experiments demonstrated the
physical association between HKc
and HKc
proteins in the apical
membrane (Fig. 8B,
lane
3). In addition, the preimmune serum
(Fig. 8D,
lane
3) and immunodepletion experiment
(Fig. 7A, lane
3) did not identify any protein
bands except immunoglobulins. These observations confirm that
HKc
protein bands are specific, and the two additional low-molecular
weight protein bands in the immunoprecipitate may be a HKc
degradation product. In contrast, an antibody to
NaK
1 identified protein in
basolateral but not in apical membranes (Fig.
8C,
lane
2). As a result, it is unlikely that
NaK
1 protein is associated with
HKc
protein or is the
-subunit for H-K-ATPase. The demonstration
of the selective increase in HKc
protein expression in apical
membrane of rat distal colon and the association of HKc
protein with
HKc
protein in apical membrane (Fig.
8B,
lane
3) strongly support the possibility
that HKc
is most likely the
-subunit for colonic H-K-ATPase
-subunit.
Although dietary K depletion unequivocally stimulates active K absorption in the large intestine of both rat (12) and mouse (25), enhancement of H-K-ATPase in dietary K depletion has been inconstant. It is generally believed that active K absorption in the distal colon is a result of an H/K exchange energized by an apical membrane H-K-ATPase (2), but the effect of dietary K depletion on colonic active K absorption and H-K-ATPase activity is complex. Confounding variables of the presumed stimulation of H-K-ATPase by dietary K depletion include its duration and the presence of two H-K-ATPase isoforms and two components of active K absorption. The two H-K-ATPases have different sensitivities to ouabain and spatial distributions (8, 31), and the two active K absorptive processes also have different sensitivities to ouabain, as well as different responsiveness to aldosterone (28). Additionally, direct demonstration of an H/K exchange in colonic apical membranes has not as yet been established, and Feldman and Ickes (10) presented evidence of a dissociation between active K absorption and protein secretion.
In conclusion, we have identified a -subunit from rat distal colon,
and its mRNA is upregulated in a tissue-specific manner in the distal
colon of K-depleted but not of Na-depleted rats. This
-subunit
protein localized in the apical membrane, physically associated with
HKc
protein. Its protein expression is selectively increased in the
apical membrane of K-depleted rats. These observations are all
consistent with the probability that HKc
is the putative
-subunit
for colonic H-K-ATPase.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Mary Guidone for excellent secretarial assistance and
Andrea Mann for excellent technical assistance in the preparation of
the HKc antibody. The
3-subunit cDNA from an
astrocytoma cell line (38) was kindly provided by Drs. T. Watanabe and
Y. Suzuki. NaK
1 antibody was
kindly provided by Dr. Michael Caplan.
![]() |
FOOTNOTES |
---|
This research was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant DK-18777. S. S. Kolla is a trainee of NIDDK Grant DK-07017.
Address for reprint requests: H. J. Binder, Yale University School of Medicine, Department of Internal Medicine, Section of Digestive Diseases, 333 Cedar St., 89 LMP, New Haven, CT 06520-8019.
Received 21 November 1997; accepted in final form 26 October 1998.
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