Am J Physiol Gastrointest Liver Physiol
281: G1369–G1377, 2001.
Colonic H⫹-K⫹-ATPase in K⫹ conservation and
electrogenic Na⫹ absorption during Na⫹ restriction
ZACHARY SPICER,1 LANE L. CLARKE,2 LARA R. GAWENIS,2 AND GARY E. SHULL1
1
Department of Molecular Genetics, Biochemistry and Microbiology, The University
of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524; and 2Department
of Biomedical Sciences, College of Veterinary Medicine and Dalton Cardiovascular
Research Center, University of Missouri, Columbia, Missouri 65211
Received 5 March 2001; accepted in final form 3 July 2001
Address for reprint requests and other correspondence: G. E.
Shull, Dept. of Molecular Genetics, Biochemistry and Microbiology,
Univ. of Cincinnati, College of Medicine, 231 Albert Sabin Way, ML
524, Cincinnati, OH 45267-0524 (E-mail: [email protected]).
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.
Atp1al1; potassium absorption; sodium absorption; hydrogen
secretion
THE COLONIC H
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⫹ ⫹
-K -ATPASE (cHKA), a member of the
P-type family of ion transport ATPases that was cloned
from distal colon (11), is expressed in apical membranes of distal colon surface cells (19, 27, 33). The
existence of an H⫹-K⫹-ATPase in distal colon, or possibly two H⫹-K⫹-ATPases (1), was first indicated by
physiological studies showing a linked pathway for H⫹
secretion and K⫹ absorption across the apical membrane of distal colon epithelial cells that was Na⫹
independent and sensitive to vanadate (12, 18, 23, 38,
39, 44). Expression studies using Xenopus oocytes demonstrated that cHKA is indeed an H⫹-K⫹-ATPase (6,
10), although there are data showing that the enzyme
can substitute Na⫹ for H⫹ and NH4⫹ for K⫹ (7–9, 32).
The role of the pump in K⫹ homeostasis has been
partially elucidated by using a gene-targeted mouse
model. cHKA⫺/⫺ mice exhibit a defect in colonic K⫹
conservation when fed either a control diet or a K⫹depleted diet (30). Dietary K⫹ restriction does not
affect cHKA mRNA, protein, or activity in colon (20,
35); however, in several models of hyperaldosteronism,
including dietary Na⫹ restriction (35), treatment with
aldosterone (20), and targeted mutation of the NHE3
Na⫹/H⫹ exchanger (36), cHKA mRNA is sharply induced.
In the rat distal colon, dietary Na⫹ restriction or
treatment with aldosterone causes a switch in Na⫹
transport from predominantly electroneutral absorption via coupled Na⫹/H⫹ and Cl⫺/HCO3⫺ exchange to
electrogenic absorption via the amiloride-sensitive epithelial Na⫹ channel (16, 31). In addition, there is a
sharp increase in both K⫹ secretion (16) and K⫹ absorption (14, 41). A K⫹-ATPase activity was suggested
as the molecular mechanism of K⫹ absorption (41) and
was hypothesized to recycle K⫹ secreted via the apical
K⫹ channel (15, 16, 42). K⫹ absorption was inhibited
by orthovanadate and ouabain (41), indicating that it is
mediated by cHKA. cHKA mRNA was induced in distal
colon of aldosterone- and dexamethasone-treated rats
(20), and cHKA mRNA, protein, and activity increased
in distal colon following dietary Na⫹ restriction (35).
The electrogenic absorption of Na⫹ from the lumen of
the colon requires either the absorption of an anion or
the secretion of a cation to maintain the appropriate
electrochemical gradient across the apical membrane.
For this reason, recycling of K⫹ that is secreted across
the apical membrane may be critical for maximum
electrogenic Na⫹ absorption via the epithelial Na⫹
channel (ENaC). Thus we hypothesized that the induction of cHKA during dietary Na⫹ restriction or other
conditions in which aldosterone levels are elevated
may allow an increased rate of K⫹ recycling across the
apical membrane, thereby facilitating Na⫹ absorption.
If so, then the loss of cHKA should impair both K⫹
Spicer, Zachary, Lane L. Clarke, Lara R. Gawenis,
and Gary E. Shull. Colonic H⫹-K⫹-ATPase in K⫹ conservation and electrogenic Na⫹ absorption during Na⫹ restriction. Am J Physiol Gastrointest Liver Physiol 281:
G1369–G1377, 2001.—Upregulation of the colonic H⫹-K⫹ATPase (cHKA) during hyperaldosteronism suggests that it
functions in both K⫹ conservation and electrogenic Na⫹ absorption in the colon when Na⫹-conserving mechanisms are
activated. To test this hypothesis, wild-type (cHKA⫹/⫹) and
cHKA-deficient (cHKA⫺/⫺) mice were fed Na⫹-replete and
Na⫹-restricted diets and their responses were analyzed. In
both genotypes, Na⫹ restriction led to reduced plasma Na⫹
and increased serum aldosterone, and mRNAs for the epithelial Na⫹ channel (ENaC) ␤- and ␥-subunits, channel-inducing factor, and cHKA were increased in distal colon. Relative
to wild-type controls, cHKA⫺/⫺ mice on a Na⫹-replete diet
had elevated fecal K⫹ excretion. Dietary Na⫹ restriction led
to increased K⫹ excretion in knockout but not in wild-type
mice. The amiloride-sensitive, ENaC-mediated short-circuit
current in distal colon was significantly reduced in knockout
mice maintained on either the Na⫹-replete or Na⫹-restricted
diet. These results demonstrate that cHKA plays an important role in K⫹ conservation during dietary Na⫹ restriction
and suggest that cHKA-mediated K⫹ recycling across the
apical membrane is required for maximum electrogenic Na⫹
absorption.
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H⫹-K⫹-ATPASE IN K⫹ CONSERVATION AND NA⫹ ABSORPTION
conservation and electrogenic Na⫹ absorption. To test
these hypotheses, we analyzed colonic Na⫹ and K⫹
conservation and electrogenic Na⫹ absorption in wildtype and cHKA⫺/⫺ mice. The results demonstrate that
the loss of cHKA impairs both K⫹ conservation and
electrogenic Na⫹ absorption during dietary Na⫹ restriction.
METHODS
Fig. 1. PCR genotyping strategy for colonic H⫹-K⫹-ATPase (cHKA)deficient mice. A: part of the wild-type cHKA allele (top) and the
mutant allele (bottom) with the neomycin resistance gene (neo)
disrupting exon 20. Arrows indicate the relative positions of the
primers used in a triplex PCR reaction that yielded 154- and 298-bp
products for the wild-type and mutant alleles, respectively (see
METHODS). B: agarose gel electrophoresis of ethidium bromidestained PCR products from wild-type (⫹/⫹), heterozygous (⫹/⫺), and
homozygous mutant (⫺/⫺) mice.
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Maintenance of mice and genotype analysis. Generation of
cHKA-deficient mice was described previously (30). For the
present study, the targeted null mutation was bred onto a
C57BL/6 background. Mice were maintained in a pathogenfree barrier facility with free access to standard mouse chow
and water until the time of the study.
Genotyping was performed by PCR analysis of genomic
DNA isolated from tail biopsies. The PCR reaction used two
cHKA gene-specific primers and one neomycin resistance
gene-specific primer. The 5⬘ cHKA primer (5⬘-CTGGAATGGACAGGCTCAACG-3⬘) in conjunction with the 3⬘ cHKA
primer (5⬘-GTACCTGAAGAGCCCCTGCTG-3⬘) amplified a
154-bp fragment of exon 20 from the wild-type allele. The 5⬘
cHKA primer and the neomycin resistance gene primer (5⬘CTGACTAGGGGAGGAGTAGAAGG-3⬘) amplified a 298-bp
fragment from the mutant allele that contains portions of
exon 20 and the neomycin resistance gene. The PCR genotyping strategy and an example of the PCR amplifications
are shown in Fig. 1.
Western blot analysis. The distal colon was removed from
two cHKA⫹/⫹ and two cHKA⫺/⫺ mice and homogenized in 2
ml sucrose buffer [5 mM Tris, pH 7.5, 0.25 M sucrose, 2 mM
EDTA, and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. A
750-␮l aliquot was centrifuged at 11,000 rpm for 30 min at
4°C. The supernatant was placed in a fresh tube and centrifuged at 50,000 rpm for 60 min at room temperature. The
membrane pellet was resuspended in buffer B (50 mM
HEPES, pH 7.6, 1 mM EDTA, and 1 mM PMSF). A portion of
the membrane preparation (10 ␮l) was separated by electrophoresis in a 10% denaturing polyacrylamide gel and transferred to a nylon membrane. The primary antibody against
cHKA (4) (a generous gift from Drs. Thomas DuBose and
Juan Codina) was incubated with the membrane at a 1:1,000
dilution for 2 h. The secondary antibody was incubated at a
1:20,000 dilution for 1 h. Detection was performed with the
SuperSignal kit (Pierce, Rockford, IL) according to the manufacturer’s protocol. The protein on the membrane was then
stained with Ponceau solution (Sigma) to confirm equal loading between lanes.
Dietary Na⫹ restriction studies. Studies were performed on
age- and sex-matched adult cHKA⫹/⫹ and cHKA⫺/⫺ mice.
Mice were fed a control diet containing 1% NaCl, followed by
a diet containing 0.01% NaCl (Harlan Teklad, Madison, WI),
and urine and feces were collected daily. Urine volume was
measured, and Na⫹ and K⫹ content were determined by
flame photometry as described previously (30) and presented
as 3-day averages. Feces from three consecutive days were
pooled, weighed, dissolved in 0.75 N nitric acid, and centrifuged. Determination of fecal Na⫹ and K⫹ content was performed by flame photometry of the supernatant as described
previously (30).
Determination of blood pH, gases, and electrolytes and
serum aldosterone. Blood (50 ␮l) was collected from the tail
vein of conscious mice and analyzed immediately for acid/
base status, blood gases, and plasma electrolytes using a
Chiron Diagnostics model 348 pH/blood gas analyzer (30).
These analyses were performed at the end of both the control
diet period and the Na⫹-restricted diet period. Serum aldosterone concentrations were also determined at the end of
each period using a 125I radioimmunoassay as previously
described (36).
Northern blot analyses. Total RNA from the kidneys of
three animals in each experimental group and total RNA
from proximal and distal colon of two animals in each group
were isolated and pooled. Total RNA (10 ␮g) was denatured,
separated by agarose gel electrophoresis, and transferred to
a nylon membrane. Hybridization was performed as described previously (36) using cDNA probes for the ␣-subunit
of cHKA; the ␤1-subunit of the Na⫹-K⫹-ATPase; channelinducing factor (CHIF); the ␣-, ␤-, and ␥-subunits of ENaC;
and the mouse L32 ribosomal protein (as a loading control).
Bioelectric measurements. Transepithelial current measurements in the distal colon of cHKA⫹/⫹ and cHKA⫺/⫺ mice
were performed under short-circuit (Isc) conditions in Ussing
chambers as described (3). Freshly isolated tissues were
mounted in voltage-clamped Ussing chambers and were
bathed in Krebs-Ringer-HCO3⫺ solution, pH 7.4 (in mM: 115
NaCl, 25 NaHCO3, 5 KCl, 1.2 MgCl2, 1.2 CaCl2, 10 serosal
glucose, and 10 mucosal mannitol). In preliminary experiments, Isc was measured both before and after the sequential
addition of 1 ␮M amiloride and then 50 ␮M amiloride to the
luminal side of the tissue. Inhibition by 1 ␮M amiloride was
only 54% of that observed with 50 ␮M amiloride. In subsequent experiments, Isc was measured both before and after
the addition of 50 ␮M amiloride.
Statistics. Statistical significance was calculated by singlefactor ANOVA, ANOVA-protected Bonferroni’s t-test, or paired
t-test as appropriate. Data are presented as means ⫾ SE.
H⫹-K⫹-ATPASE IN K⫹ CONSERVATION AND NA⫹ ABSORPTION
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RESULTS
Fig. 2. Western blot analysis of distal colon membranes from Na⫹restricted cHKA⫹/⫹ and cHKA⫺/⫺ mice. Membranes were isolated
from distal colon of 2 cHKA⫹/⫹ and 2 cHKA⫺/⫺ mice, separated by
denaturing polyacrylamide gel electrophoresis, and probed with a
cHKA-specific antibody. A single band, corresponding in size to that
of the colonic H⫹-K⫹-ATPase, was clearly present only in the
cHKA⫹/⫹ distal colon.
AJP-Gastrointest Liver Physiol • VOL
Fig. 3. Urinary K⫹ (A) and Na⫹ (B) excretion for cHKA⫹/⫹ and
cHKA⫺/⫺ mice. Animals were housed in metabolic cages and fed a 1%
NaCl diet for 6 days (days 1–6), followed by a 0.01% NaCl diet for 12
days (days 7–18) (n ⫽ 9 mice for each genotype). Urine was collected,
and K⫹ content and Na⫹ content were analyzed by flame photometry. Results are expressed as total K⫹ or Na⫹ excreted per 3 days.
Values are means ⫾ SE.
Urinary and fecal excretion of K⫹ and Na⫹. As shown
in Fig. 3A, urinary K⫹ excretion was similar for Na⫹replete cHKA⫹/⫹ (1,199 ⫾ 113 ␮mol/3 days) and
cHKA⫺/⫺ (1,143 ⫾ 89 ␮mol/3 days) mice but differed
significantly following Na⫹ restriction (932 ⫾ 88 and
754 ⫾ 75 ␮mol/3 days, respectively; P ⬍ 0.01). Urinary
Na⫹ excretion (Fig. 3B) was similar for cHKA⫹/⫹ and
cHKA⫺/⫺ mice during the control period (734 ⫾ 122
and 855 ⫾ 132 ␮mol/3 days, respectively) but decreased by ⬃99%, to 7.46 ⫾ 0.85 and 6.96 ⫾ 1.57
␮mol/3 days, respectively, following Na⫹ restriction.
The quantity of feces excreted by cHKA⫹/⫹ and
cHKA⫺/⫺ mice did not differ significantly during the
control (1.83 ⫾ 0.06 and 2.11 ⫾ 0.19 g/3 days, respectively) or Na⫹-restricted periods (2.21 ⫾ 0.16 and
2.06 ⫾ 0.13 g/3 days, respectively). As observed previously (30), fecal K⫹ excretion (Fig. 4A) was greater for
cHKA⫺/⫺ mice than for cHKA⫹/⫹ mice during the control diet period (379 ⫾ 23 and 248 ⫾ 16 ␮mol/3 days,
respectively; P ⬍ 0.001). When fed a Na⫹-restricted
diet, cHKA⫹/⫹ mice maintained the same level of fecal
K⫹ excretion as during the control period. In contrast,
during the last 3 days of Na⫹ restriction, fecal K⫹
excretion by cHKA⫺/⫺ mice was 2.8-fold greater than
that of wild-type mice (693 ⫾ 38 and 247 ⫾ 24 ␮mol/3
days, respectively; P ⬍ 0.001). Fecal excretion of Na⫹
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Dietary Na⫹ restriction was carried out using wildtype and cHKA⫺/⫺ mice, which were housed individually in metabolic cages. The mice received food containing 1% NaCl during the 6-day control period (days 1–6)
and 0.01% NaCl during the 12-day period of Na⫹ restriction (days 7–18). Urine and feces were collected
and analyzed, and at the conclusion of each dietary
period, blood was collected for analysis of blood gases,
pH, plasma electrolytes, and serum aldosterone.
Northern blot analyses of kidney and intestinal segments of Na⫹-replete and Na⫹-restricted mice were
performed, and a separate group of Na⫹-replete and
Na⫹-restricted mice were used for analysis of Isc in
distal colon.
Body weights. At the beginning of the study, cHKA⫹/⫹
and cHKA⫺/⫺ mice had similar body weights (23.8 ⫾
1.5 and 23.2 ⫾ 1.1 g, respectively). The mice were also
weighed at the end of the control diet period (day 6:
cHKA⫹/⫹, 23.2 ⫾ 1.1 g; cHKA⫺/⫺, 22.7 ⫾ 1.1 g), at the
midpoint of the Na⫹-restricted period (day 12: cHKA⫹/⫹,
23.8 ⫾ 1.3 g; cHKA⫺/⫺, 22.7 ⫾ 1.1 g), and at the end of
the experiment (day 18: cHKA⫹/⫹, 24.4 ⫾ 1.4 g;
cHKA⫺/⫺, 23.3 ⫾ 1.1 g). There were no significant differences in body weight for either genotype during the
18-day study, nor were there any significant differences
in body weight between genotypes at any time point.
Western blot analysis of cHKA protein in distal colon
of wild-type and cHKA⫺/⫺⫹mice. To confirm the effectiveness of the gene-targeting strategy, membranes
were isolated from the distal colons of Na⫹-restricted
wild-type and cHKA⫺/⫺ mice and examined by Western
blot analysis. An antibody directed against cHKA identified a ⬃110-kDa protein in cHKA⫹/⫹ distal colon, but
only a trace band was seen in the same position in
cHKA⫺/⫺ distal colon (Fig. 2). The faint band seen in
the knockout lanes may have been due to nonspecific
hybridization, because it corresponds in position to a
strong protein band observed in all lanes after staining
with Ponceau solution (not shown). Alternatively, it
may represent trace levels of a mutant protein, lacking
some of the COOH terminal transmembrane domains,
that could be produced from the aberrant cHKA transcripts.
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H⫹-K⫹-ATPASE IN K⫹ CONSERVATION AND NA⫹ ABSORPTION
Table 1. Serum electrolytes and acid-base status
of cHKA⫹/⫹ and cHKA⫺/⫺ mice on 1% and
0.01% NaCl diets
1%
⫹/⫹
0.01%
⫺/⫺
⫹/⫹
⫺/⫺
149.1 ⫾ 0.5 150.0 ⫾ 0.6 146.8 ⫾ 0.7* 146.9 ⫾ 0.4**
Na , mM
K⫹, mM
6.49 ⫾ 0.42 6.17 ⫾ 0.16 6.02 ⫾ 0.20 6.82 ⫾ 0.49
Cl⫺, mM
113.4 ⫾ 0.7 113.9 ⫾ 0.83 117.1 ⫾ 0.9 112.0 ⫾ 1.2
pH
7.45 ⫾ 0.01 7.45 ⫾ 0.01 7.49 ⫾ 0.01 7.46 ⫾ 0.01
HCO3⫺, mM 24.2 ⫾ 1.3
24.4 ⫾ 1.1
24.4 ⫾ 0.7
22.5 ⫾ 0.8
⫹
Values are means ⫾ SE for 8 mice in each group. cHKA, colonic
H⫹-K⫹-ATPase. * P ⬍ 0.05 and ** P ⬍ 0.01 for differences between
diets within the same genotype.
(Fig. 4B) was similar for cHKA⫺/⫺ and cHKA⫹/⫹ mice
during the control period (249 ⫾ 21 and 217 ⫾ 19
␮mol/3 days, respectively). When fed a Na⫹-restricted
diet, fecal Na⫹ excretion decreased by ⬎90% in both
genotypes, although it was slightly greater in knockout
than in wild-type mice (11.0 ⫾ 0.5 and 13.7 ⫾ 1.3
␮mol/3 days, respectively; P ⫽ 0.023).
Plasma electrolytes and serum aldosterone. Plasma
electrolytes and systemic acid/base status were analyzed at the end of each dietary period (Table 1). Serum
Na⫹ concentrations in Na⫹-replete cHKA⫹/⫹ and
cHKA⫺/⫺ mice were 149.1 ⫾ 0.5 mM and 150.0 ⫾ 0.6
mM, respectively. Serum Na⫹ did not differ significantly between the two genotypes on either diet; however, both groups of mice became hyponatremic after
12 days of Na⫹ restriction, with serum Na⫹ of 146.8 ⫾
0.7 mM in wild-type mice (P ⫽ 0.034) and 146.9 ⫾ 0.4
mM in the knockout (P ⬍ 0.01). Serum Cl⫺ concentrations did not differ significantly between cHKA⫹/⫹ and
cHKA⫺/⫺ mice on a Na⫹-replete diet. During Na⫹ restriction, serum Cl⫺ did not change in the knockout
mice but was slightly elevated in wild-type mice (P ⫽
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Fig. 5. Serum aldosterone concentrations for cHKA⫹/⫹ and cHKA⫺/⫺
mice after being fed a diet containing 1% NaCl (Normal) for 6 days
and then a diet containing 0.01% NaCl (Restricted) for 12 days (n ⫽
6 wild-type and 5 knockout mice). Values are means ⫾ SE. *P ⬍ 0.01
between normal and Na⫹-restricted diets within each genotype;
there were no significant differences between the 2 genotypes.
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Fig. 4. Fecal K⫹ and Na⫹ excretion for cHKA⫹/⫹ and cHKA⫺/⫺ mice.
Animals were fed a 1% NaCl diet (days 1–6) followed by a 0.01%
NaCl diet (days 7–18) (n ⫽ 13 for each genotype; this includes the 9
pairs in Fig. 3 and 4 additional pairs). Feces were pooled at 3-day
intervals and were analyzed for K⫹ and Na⫹ content. A: total K⫹
excreted per 3 days for cHKA⫹/⫹ and cHKA⫺/⫺ mice. All values for
cHKA⫺/⫺ mice are significantly greater (P ⬍ 0.001) than the corresponding values for cHKA⫹/⫹ mice. †P ⬍ 0.01 between K⫹ excreted
on days 1–3 and K⫹ excreted on the indicated days for cHKA⫺/⫺ mice
only. B: total Na⫹ excreted per 3 days for cHKA⫹/⫹ and cHKA⫺/⫺
mice. Values are means ⫾ SE.
0.024; this is most likely a statistical anomaly). Serum
K⫹, pH, and HCO3⫺ concentrations were essentially the
same in both genotypes and were unaffected by diet.
Serum aldosterone levels (Fig. 5) were similar in
wild-type and cHKA⫺/⫺ mice (1,460 ⫾ 204 pg/ml and
1,112 ⫾ 101 pg/ml, respectively) at the end of the
control diet period. After 12 days of Na⫹ restriction,
serum aldosterone increased approximately threefold
in both cHKA⫹/⫹ and cHKA⫺/⫺ mice, to 4,333 ⫾ 312
pg/ml and 3,524 ⫾ 597 pg/ml, respectively.
Northern blot analysis of kidneys and colon. Northern blot hybridization was performed using kidney
RNA from Na⫹-replete and Na⫹-restricted mice (Fig. 6,
left). A previous study showed that cHKA mRNA is
expressed at almost undetectable levels in kidneys of
K⫹-replete wild-type mice but is sharply induced by
K⫹-depletion (30). However, we observed no signal for
cHKA mRNA in kidneys of Na⫹-replete mice of either
genotype, and there was no evidence of cHKA induction in response to Na⫹ restriction. Na⫹ restriction had
no apparent effect on kidney mRNA levels for the
ENaC subunits, CHIF, or the Na⫹-K⫹-ATPase ␤1-subunit.
Northern blot analyses of RNA from proximal and
distal colon revealed similar changes in both genotypes
H⫹-K⫹-ATPASE IN K⫹ CONSERVATION AND NA⫹ ABSORPTION
in response to Na⫹ restriction (Fig. 6, right). cHKA
mRNA was not detected in proximal colon of Na⫹replete mice of either genotype, but very low levels
could be detected after Na⫹ restriction (Fig. 6; longer
exposures not shown). In distal colon, expression of
both the wild-type cHKA mRNA in cHKA⫹/⫹ mice and
the mutant mRNA (which is larger due to insertion of
the neomycin resistance gene) in cHKA⫺/⫺ mice were
induced after Na⫹ restriction. Na⫹-K⫹-ATPase ␤1-subunit mRNA increased slightly in both proximal and
distal colon of cHKA⫹/⫹ and cHKA⫺/⫺ mice after Na⫹
restriction, whereas mRNA for the ␣-subunit of ENaC
was unchanged. In Na⫹-replete mice, CHIF mRNA
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was not detected in proximal colon, and with the short
(3 h) exposure time shown in Fig. 6, CHIF mRNA was
detected at only trace levels in distal colon. In contrast,
it was sharply induced in distal colon of both genotypes
after Na⫹ restriction. Na⫹ restriction also caused a
sharp induction of the mRNAs encoding the ␤- and
␥-subunits of ENaC in proximal and distal colon of both
groups of mice.
Isc in the distal colon of Na⫹-restricted cHKA⫹/⫹ and
cHKA⫺/⫺ mice. In an initial set of experiments designed to test the concentrations of amiloride needed to
fully inhibit ENaC, we measured transepithelial current in the distal colon of Na⫹-restricted cHKA⫹/⫹ and
cHKA⫺/⫺ mice under short-circuit conditions in the
absence of amiloride and then in the presence of 1 ␮M
and then 50 ␮M amiloride. Dietary Na⫹ restriction was
used to enhance the magnitude of the ENaC-mediated
currents. The basal Isc in cHKA⫺/⫺ colon was significantly lower than that in cHKA⫹/⫹ colon (⫺39.1 ⫾ 6.7
␮A/cm2 and ⫺193.2 ⫾ 14.1 ␮A/cm2, respectively; P ⬍
0.01; Fig. 7). After the luminal addition of 1 ␮M amiloride, the Isc decreased in both groups (cHKA⫺/⫺,
⫺13.7 ⫾ 3.3 ␮A/cm2; cHKA⫹/⫹, ⫺59.7 ⫾ 6.9 ␮A/cm2;
P ⬍ 0.01). Addition of 50 ␮M amiloride led to a further
reduction in the Isc (cHKA⫺/⫺, 0.5 ⫾ 1.2 ␮A/cm2;
cHKA⫹/⫹, 22.7 ⫾ 4.1 ␮A/cm2; P ⬍ 0.01). In subsequent
experiments, 50 ␮M amiloride was used to inhibit the
ENaC-mediated current.
In the second set of experiments, transepithelial
currents were measured in the distal colon of both
Na⫹-replete and Na⫹-restricted cHKA⫹/⫹ mice and
cHKA⫺/⫺ mice. The basal Isc did not differ significantly
between Na⫹-replete cHKA⫺/⫺ and cHKA⫹/⫹ mice
(⫺22.9 ⫾ 3.5 and ⫺35.6 ⫾ 7.6 ␮A/cm2, respectively);
however, after the luminal addition of 50 ␮M amiloride, the Isc differed significantly between the two
groups (cHKA⫺/⫺, ⫺9.2 ⫾ 1.6 ␮A/cm2; cHKA⫹/⫹, 5.9 ⫾
2.6 ␮A/cm2; P ⬍ 0.01) (Fig. 8A). The amiloride-sensitive Isc, representing the contribution from ENaC, was
approximately threefold greater in Na⫹-replete wildtype distal colon than in that of the mutant (41.5 ⫾ 7.2
and 13.7 ⫾ 3.3 ␮A/cm2, respectively; P ⬍ 0.01; Fig. 8B).
Compared with that observed under Na⫹-replete conditions, the basal Isc measured under conditions of
Fig. 7. Effects of amiloride on short-circuit current (Isc) in the distal
colon of Na⫹-restricted cHKA⫹/⫹ and cHKA⫺/⫺ mice. Isc under basal
conditions and after the sequential addition of 1 ␮M and 50 ␮M
amiloride are shown (n ⫽ 6 cHKA⫹/⫹ and 5 cHKA⫺/⫺ mice). Values
are means ⫾ SE. *P ⬍ 0.01 between cHKA⫹/⫹ and cHKA⫺/⫺ within
each treatment. †P ⬍ 0.01 for difference from basal current of same
genotype.
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Fig. 6. Northern blot analysis of RNA from kidney, proximal colon,
and distal colon of cHKA⫹/⫹ and cHKA⫺/⫺ mice maintained on
normal Na⫹-replete (N) or Na⫹-restricted (R) diets. For each genotype and dietary group, total RNA was isolated and pooled from the
kidneys of 3 mice and from the proximal and distal colons of 2 mice.
Each lane contained 10 ␮g of total RNA. The probes were: ␣-subunit
of cHKA; ␤1 subunit of Na⫹-K⫹-ATPase (␤1NKA); channel-inducing
factor (CHIF); ␣-, ␤-, and ␥-subunits of the epithelial Na⫹ channel
(␣ENaC, ␤ENaC, and ␥ENaC); and the L32 ribosomal protein (L32)
as a loading control.
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H⫹-K⫹-ATPASE IN K⫹ CONSERVATION AND NA⫹ ABSORPTION
dietary Na⫹ restriction increased ⬃4.9-fold in
cHKA⫹/⫹ mice (to ⫺173.0 ⫾ 13.7 ␮A/cm2; P ⬍ 0.01) and
increased only 2.2-fold in cHKA⫺/⫺ mice (to ⫺50.2 ⫾
7.7 ␮A/cm2; P ⬍ 0.01) (Fig. 8A). After the luminal
addition of 50 ␮M amiloride, the Isc did not differ
significantly between cHKA⫺/⫺ and cHKA⫹/⫹ mice
(⫺7.6 ⫾ 3.5 and 8.4 ⫾ 7.2 ␮A/cm2, respectively). The
amiloride-sensitive Isc was ⬃4.3-fold greater in Na⫹restricted cHKA⫹/⫹ distal colon than in that of the
mutant (181.5 ⫾ 14.5 and 42.6 ⫾ 6.8 ␮A/cm2, respectively; P ⬍ 0.01; Fig. 8B).
DISCUSSION
Studies with cHKA⫺/⫺ mice have shown that the
cHKA plays an important role in K⫹ conservation in
the colon during dietary K⫹ restriction (30), although
its mRNA is not induced in the colon under these
conditions (35). K⫹ restriction and the resulting low
serum aldosterone levels cause a reduction in apical
K⫹ secretion; hence, there is much less K⫹ to recover
and normal levels of the pump are apparently sufficient to reduce fecal K⫹ losses to very low levels (30). In
contrast, cHKA mRNA, protein, and activity in distal
colon are increased in response to dietary Na⫹ depriAJP-Gastrointest Liver Physiol • VOL
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Fig. 8. Isc in the distal colon of cHKA⫹/⫹ and cHKA⫺/⫺ mice after
being fed a 1% NaCl diet (normal) or a 0.01% NaCl diet (restricted)
for 12 days. A: Isc under basal conditions and after the addition of
amiloride. B: change in Isc after the addition of amiloride (n ⫽ 8
Na⫹-replete cHKA⫹/⫹ mice, 8 Na⫹-replete cHKA⫺/⫺ mice, 12 Na⫹restricted cHKA⫹/⫹ mice, and 11 Na⫹-restricted cHKA⫺/⫺ mice; the
Na⫹- restricted mice include the 6 cHKA⫹/⫹ and 5 cHKA⫺/⫺ mice
analyzed in Fig. 7). Values are means ⫾ SE. *P ⬍ 0.01 between
cHKA⫹/⫹ and cHKA⫺/⫺. †P ⬍ 0.01 between 1% NaCl diet and 0.01%
NaCl diet.
vation or elevated serum aldosterone levels (20, 35),
suggesting that its activity might be particularly important under conditions in which electrogenic Na⫹
absorption predominates in the colon (22). In the current study, we used cHKA knockout mice to examine
the role of the pump in K⫹ conservation during dietary
Na⫹ restriction and to determine whether its absence
affects the rate of electrogenic Na⫹ absorption.
Dietary Na⫹ restriction or treatment with aldosterone has been shown to stimulate NaCl absorption in
the rat proximal colon via coupled electroneutral
Na⫹/H⫹ and Cl⫺/HCO3⫺ exchange (16). In the rat distal
colon, however, aldosterone reduces electroneutral
transport of Na⫹ and stimulates electrogenic transport
via ENaC, the amiloride-sensitive Na⫹ channel (16).
To maintain the membrane potential needed for electrogenic absorption of Na⫹, aldosterone stimulates secretion of K⫹ through a Ba2⫹-sensitive K⫹ channel
(42). Thus it seemed likely that a major function of
cHKA in the distal colon of Na⫹-restricted mice would
be the conservation of K⫹ secreted through apical K⫹
channels; however, an additional function also seemed
likely. Secretion of K⫹ and electrogenic Na⫹ absorption
have been shown to be dependent on the Na⫹-K⫹ATPase and Na⫹-K⫹-2Cl⫺ cotransporter (42), both of
which mediate K⫹ uptake across the basolateral membrane. Nevertheless, it was reasonable to anticipate
that recycling of secreted K⫹ via the colonic H⫹-K⫹ATPase, which would be expected to enhance the apical
K⫹ gradient, might also be required for maximum
electrogenic Na⫹ absorption.
Neither cHKA⫹/⫹ nor cHKA⫺/⫺ mice fed a 0.01%
NaCl diet exhibited overt pathology, morbidity, or loss
of body weight. Serum K⫹, Cl⫺, and HCO3⫺ concentrations and serum pH were not affected by Na⫹ restriction, but both genotypes exhibited a mild reduction in
plasma Na⫹ and elevated serum aldosterone levels by
the end of the Na⫹ restriction period. In the distal
colon, Na⫹ restriction induced cHKA mRNA, and in
both proximal and distal colon it caused a slight induction of mRNA for the Na⫹-K⫹-ATPase ␤1-subunit,
which has been shown to associate with the cHKA
␣-subunit in vivo (4, 26). CHIF, which activates a K⫹
current (2), was induced only in distal colon. In both
proximal and distal colon, ENaC mRNAs were expressed at similar levels in Na⫹-replete cHKA⫹/⫹ and
cHKA⫺/⫺ mice, and ENaC ␤- and ␥-subunit mRNAs
were sharply induced in both segments during Na⫹
restriction. These results demonstrated that dietary
Na⫹ restriction in the mouse had the expected effects
on Na⫹ homeostasis and serum aldosterone and that it
modified ion transporter mRNA expression in cHKA⫹/⫹
and cHKA⫺/⫺ colon in a manner similar to that reported for rat colon (2, 13, 20, 35, 43).
When fed a control diet containing 1% NaCl, the K⫹
content of the feces of cHKA⫺/⫺ mice was ⬃50% higher
than for cHKA⫹/⫹ mice. During the Na⫹ restriction
period, wild-type mice maintained the same fecal K⫹
levels as observed on the Na⫹-replete diet, but the fecal
K⫹ content of the knockout increased to a level 2.8-fold
greater than that of wild-type mice. These data provide
H⫹-K⫹-ATPASE IN K⫹ CONSERVATION AND NA⫹ ABSORPTION
AJP-Gastrointest Liver Physiol • VOL
distal colon, it was surprising that cHKA⫺/⫺ mice exhibited only a mild increase in fecal Na⫹ excretion,
even when maintained on a Na⫹-depleted diet. A possible explanation for this discrepancy is that apical
Na⫹/H⫹ exchange activity is higher in cHKA⫺/⫺ distal
colon than in wild-type distal colon, thereby compensating for the reduction in ENaC activity. Our measurements of ENaC activity do not allow an estimation
of the relative amounts of Na⫹ being absorbed via the
electroneutral process of coupled Na⫹/H⫹ and Cl⫺/
HCO3⫺ exchange, electrogenic transport via ENaC, or
other processes that have not yet been well characterized. Similarly, the relative amounts of K⫹ being absorbed by cHKA or other K⫹-recovery mechanisms,
such as the putative second H⫹-K⫹-ATPase (1), is unclear. An additional uncertainty is the physiological
function of the apparent Na⫹ and NH4⫹ ATPase and
transport activity that has been attributed to cHKA or
the second putative H⫹-K⫹-ATPase (7–9, 32). In the
studies described here, we have considered only the
functions of cHKA operating in an H⫹/K⫹ exchange
mode. In future studies, it will be important to develop
a quantitative understanding of the relative contributions of the various apical transport mechanisms and
to understand the physiological functions and mechanistic basis for the putative Na⫹/K⫹ and H⫹/NH4⫹ exchange modes of the colonic H⫹-K⫹-ATPase (7–9). Interestingly, CHIF is related to the Na⫹-K⫹-ATPase
␥-subunit (40), raising the intriguing possibility that it
might interact with and affect the activity of cHKA.
The conditions under which CHIF is induced, such as
during dietary Na⫹ depletion or in diarrheal states
Fig. 9. Proposed model of transport mechanisms mediating transepithelial KCl and NaCl absorption in colonic surface cells. KCl absorption occurs by coupled Cl⫺/HCO3⫺ and H⫹/K⫹ exchange (via cHKA)
on the apical membrane and by KCl cotransport (34) and possibly K⫹
and Cl⫺ channel activity on the basolateral membrane. Under Na⫹replete conditions, NaCl absorption occurs largely via coupled
Na⫹/H⫹ and Cl⫺/HCO3⫺ exchange, whereas during dietary Na⫹ restriction Na⫹ is absorbed electrogenically via ENaC (16, 31). The
appropriate electrochemical gradients for Na⫹ absorption are maintained by K⫹ secretion via an apical membrane channel (14, 42), K⫹
recycling and H⫹ secretion by cHKA (38, 39, 41), and the activities of
the Na⫹-K⫹-ATPase and Na⫹-K⫹-2Cl⫺ cotransporter (42) on the
basolateral membrane.
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direct confirmation of the hypothesis that cHKA plays
an important role in colonic K⫹ conservation not only
during dietary K⫹ restriction, as shown previously
(30), but also during dietary Na⫹ restriction. This suggests that part of the biological rationale for the induction of cHKA in colon by Na⫹ restriction or under
conditions in which serum aldosterone is elevated, as
observed previously in rat and mouse colon (20, 35, 36),
is to recover the K⫹ being secreted under these conditions (16).
The Na⫹ content of the feces of both cHKA⫹/⫹ and
cHKA⫺/⫺ mice decreased by ⬃90% during the Na⫹
restriction period, although it remained slightly higher
in cHKA⫺/⫺ mice than in cHKA⫹/⫹ mice. To analyze
Na⫹ transport in the distal colon of cHKA⫹/⫹ and
cHKA⫺/⫺ mice, Isc in colons of Na⫹-replete and Na⫹restricted mice were analyzed. These experiments
showed that the amiloride-sensitive Na⫹ current in
both the Na⫹-replete and Na⫹-restricted cHKA⫺/⫺ colon was substantially less than that observed in the
corresponding cHKA⫹/⫹ colon. Although ENaC activity
was induced by Na⫹ depletion in both genotypes, the
Na⫹ current present in Na⫹-restricted cHKA⫺/⫺ colon
was essentially the same as that observed in Na⫹replete cHKA⫹/⫹ colon and ⬃25% of that in Na⫹-restricted cHKA⫹/⫹ colon (see Fig. 8). But why would the
loss of cHKA cause a reduction in electrogenic Na⫹
absorption? One possibility, as suggested by others
(22), is that cHKA functions as a K⫹-recycling mechanism to maintain K⫹ secretion and a favorable membrane potential for electrogenic Na⫹ absorption. Similar ion recycling mechanisms have been observed in
other systems. For example, gene knockout studies
showed that a reduction in the levels of the Na⫹-K⫹ATPase ␣2-isoform, but not the ␣1-isoform, enhances
cardiac contractility and Ca2⫹ loading of cardiac myocytes (20). It was concluded that regulation of subsarcolemmal Na⫹ concentrations by the ␣2-isoform controls the activity of the Na⫹/Ca2⫹ exchanger, with the
resulting alteration in intracellular Ca2⫹ affecting cardiac contractility (21). In another recent example, the
extrusion of H⫹ via an apical Na⫹/H⫹ exchanger was
shown to reduce the subapical H⫹ concentration in
colonocytes, thereby creating a pH microclimate that
stimulated the uptake of short-chain fatty acids (17).
Finally, it is well established that recycling of K⫹ by
the renal outer medullary K⫹ channel is critical for
activity of the apical Na⫹-K⫹-2Cl⫺ cotransporter of the
thick ascending limb and for accompanying paracellular transport of Na⫹ (37). The results of the present
study indicate that K⫹ absorption by the colonic H⫹K⫹-ATPase reduces luminal K⫹ concentrations and are
consistent with the possibility that it serves as a K⫹loading mechanism that increases subapical K⫹ concentrations. If this hypothesis is correct, then the resulting transmembrane K⫹ gradient would enhance
electrogenic K⫹ secretion, thereby contributing to the
electrical potential needed for maximum electrogenic
Na⫹ absorption.
Given that ENaC-mediated Na⫹ currents in
cHKA⫺/⫺ distal colon were much less than in wild-type
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AJP-Gastrointest Liver Physiol • VOL
We thank Drs. Thomas DuBose and Juan Codina for the generous
gift of the cHKA antibody used in this study.
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-50594 and DK-48816.
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such as in the NHE3-deficient mouse, are those in
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Although our primary objective was to investigate
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Na⫹ absorption in the colon, we were also interested in
determining whether dietary Na⫹ restriction might
affect renal Na⫹ handling. cHKA is expressed at very
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