From the Departments of Internal Medicine and of Integrative Biology, Pharmacology and Physiology, The University of Texas Medical School, Houston, Texas 77030
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ABSTRACT |
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The H+-K+-ATPase of
renal collecting duct mediates K+ conservation during
chronic hypokalemia. K+ deprivation promotes
H+-K+-ATPase 2 (HK
2) gene expression in
the medullary collecting duct, the principal site of active
K+ reabsorption, suggesting that this isozyme contributes
to renal K+ reclamation. We report here that alternative
transcriptional initiation and mRNA splicing give rise to distinct
N-terminal variants of the HK
2 subunit. Sequence analysis and
in vitro translation revealed that HK
2a corresponds to
the known HK
2 cDNA (Crowson, M. S., and Shull, G. E. (1992) J. Biol. Chem. 267, 13740-13748), whereas
HK
2b represents a novel variant truncated by 108 amino acids at its
N terminus. HK
2b mRNA contains a complex 5
-untranslated region
with eight upstream open reading frames, features implicated in
translational regulation of other genes. Heterologous expression of
HK
2b with and without the gastric
H+-K+-ATPase
subunit in HEK 293 cells
indicated that this variant encodes a K+ uptake mechanism
that is relatively Sch 28080-resistant, partially sensitive to ouabain,
and appears to require coexpression with the gastric
H+-K+-ATPase
subunit for optimal functional
activity. Northern analysis demonstrated that both subtypes
(HK
2b > HK
2a) are expressed abundantly in distal colon and
modestly in proximal colon and kidney. Moreover, the abundance of the
two mRNAs increases coordinately among the renal zones, but not in
colon, with chronic K+ deprivation. These results
demonstrate the potential for complex control of HK
2 gene expression
by transcriptional and posttranscriptional mechanisms not recognized in
other members of the
Na+-K+-ATPase/H+-K+-ATPase
family.
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INTRODUCTION |
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The maintenance of body potassium (K+) balance is critical to the normal function of all cells. Perturbations in K+ homeostasis disrupt normal cell growth and division, metabolism, volume and osmotic regulation, acid-base economy, and the excitability of nerve and contractile cells. The kidney is the principal arbiter of body K+ balance in mammals, adjusting K+ excretion to match large variations in dietary K+ intake. The late distal tubule and collecting duct have the dual ability to secrete and reabsorb K+ as needed to effect this balance (1, 2). In response to chronic dietary K+ deprivation, these segments, in particular the OMCD1 actively reclaim filtered K+. Physiological, biochemical, and molecular biological studies have shown that this adaptation is principally attributable to increased expression and/or activity of an H+-K+-ATPase in the luminal membrane of these segments (2-9). A similar transport system(s) has been identified in the apical membrane of mammalian distal colon, where it, too, effects active K+ absorption (10, 11). Although active K+ absorption in the distal colon is enhanced during K+ depletion (11) and participates to a limited degree in restoring K+ balance, the identity of the specific K+-ATPase that is up-regulated remains controversial (12).
The H+-K+-ATPases constitute a subfamily of
isozymes that belong to the
X+-K+-ATPase multigene family, which
also includes the Na+-K+-ATPase isoforms. The
X+-K+-ATPases share common catalytic
and ion transport mechanisms and an apparent requirement for
heterodimeric (:
) structure. The X+-K+-ATPase
subunits exhibit
considerable (~65%) structural homology and contribute most of the
functional properties of the holoenzymes, but they can be distinguished
to a degree from one another on the basis of organ distributions and
sensitivities to the inhibitors ouabain and Sch 28080 (13). To date,
three distinct H+-K+-ATPase
subunits have
been cloned from mammals, structurally characterized, and expressed in
heterologous systems. The H+-K+-ATPase
1
subunit (HK
1) was first cloned from and is principally expressed in
stomach (14), where it plays a major role in gastric acid secretion.
Messenger RNA encoding this gene was also identified in the renal
collecting duct (3). The pharmacological signature of the HK
1
protein is its high sensitivity to inhibition by Sch 28080 and its
complete resistance to inhibition by ouabain (15, 16). The
H+-K+-ATPase
2 cDNA was first cloned
from rat distal colon (17), where it is abundantly expressed, and lower
levels of HK
2 mRNA were reported in proximal colon (17), uterus
(17), and kidney (5-8). Expression of the HK
2 subunit with the
known rat X+-K+-ATPase
subunits
(16) or toad bladder H+-K+-ATPase
subunit
(18) in Xenopus laevis oocytes resulted in the appearance of
active H+-K+ exchange that was virtually
resistant to Sch 28080 and partially inhibited by ouabain. When HK
2
was expressed without an exogenous
subunit in Sf9 cells, the
resultant K+-ATPase activity was Sch 28080- and
ouabain-resistant (19). A third H+-K+-ATPase
subunit cDNA, termed ATP1AL1 (or
H+-K+-ATPase
4), was cloned from a human
skin cDNA library (20), and transcripts encoding this gene product
were also detected in human brain and kidney but not colon (20).
Coexpression of the ATP1AL1 subunit and the rabbit gastric
H+-K+-ATPase
subunit (HK
g)
in Xenopus oocytes (21) or HEK 293 cells (22) resulted in
the expression of functional H+-K+ pumps that
were partially sensitive to both Sch 28080 and ouabain.
Recent studies by our laboratory and others have shown that chronic
K+ deprivation enhances HK2, but not HK
1 (3), gene
expression in the OMCD (5-8) and proximal portion of the inner
medullary collecting duct (6) of rats. In one of these studies (8), HK
2 protein levels, but not mRNA levels, were enhanced in the outer medulla of K+-deprived rats, suggesting the potential
operation of translational or post-translational control mechanisms. In
contrast to kidney, chronic hypokalemia does not appear to alter HK
2
mRNA (5, 8) or protein (8) abundance in rat distal colon. Moreover, recent work demonstrating disparate effects of adrenalectomy, dexamethasone treatment (5), and dietary Na+ depletion (8)
on HK
2 abundance in the rat outer medulla and distal colon indicated
that cell type-specific regulatory mechanisms govern HK
2 gene
expression in these tissues.
Since both transcriptional and translational control mechanisms, as
well as alternative mRNA splicing, can lead to regulated, tissue-specific gene expression, we hypothesized that these mechanisms might operate to confer structural and/or regulatory diversity to the
HK2 subunit gene. Although the structural organization of the rat
and human HK
1 (23) and human ATP1AL1 (24) genes is known,
that of the rat HK
2 gene has not been described. We report here that
distinct transcription initiation sites in the rat HK
2 gene and
alternative mRNA splicing, combined regulatory mechanisms not known
to be utilized by other members of the
X+-K+-ATPase
subunit family,
direct the synthesis of two N-terminal HK
2 variants that are
expressed principally, if not exclusively, in the kidney and colon and
that appear to respond coordinately in kidney to chronic K+
deprivation.
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EXPERIMENTAL PROCEDURES |
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Animal Protocols-- Male Sprague-Dawley rats (180-220 g) were fed normal rat chow (150 mEq KCl/kg chow, TD88081, Harlan Teklad) or a nominally K+-free (TD88082, Harlan Teklad) diet for 2 weeks. This K+-restriction protocol reproducibly results in significant hypokalemia (3) and has been used in our previous studies (3, 6).
Oligonucleotide Primers--
PCR primers not included in
specific kits were synthesized by Genosys, Inc. (The Woodlands, TX).
The sequences of the various HK2 subunit primers are presented in
Fig. 1A, and those of the HK
g subunit are
given below.
5-RACE and Cloning of H+-K+-ATPase
2b
cDNA--
The 5
-RACE protocol was performed using the
MarathonTM cDNA Amplification Kit
(CLONTECH, Palo Alto, CA), according to the
manufacturer's instructions. First strand cDNAs were generated
from 1 µg of rat kidney poly(A)+ RNA, using Moloney
murine leukemia virus reverse transcriptase and a modified locking
oligo(dT) primer containing two degenerate nucleotide positions at its
3
end provided with the kit. Second strand synthesis was accomplished
with a mixture of Escherichia coli DNA polymerase I, RNase
H, and E. coli DNA ligase. After creation of blunt ends with
T4 DNA polymerase, the double-stranded cDNA was ligated to adapter
primer 1 furnished with the kit, using T4 DNA ligase. The
anchor-ligated cDNAs were then subjected to 5
-RACE using a nested
primer (adapter primer 2, supplied with the RACE kit) complementary to
adapter primer 1, HK
2-specific reverse primer (P1, Fig.
1A) complementary to nucleotides +344 to +325 of the
published HK
2 cDNA sequence (17), and the components of the
AdvantageTM cDNA Amplification Kit
(CLONTECH). PCR cycling conditions were as follows:
94 °C × 1 min, followed by 28 cycles of 94 °C × 30 s, 68 °C × 4 min, and a final step of 68 °C × 4 min. Ten µl of the amplified products were separated by
electrophoresis in a 1% agarose gel and visualized by ethidium bromide
staining and UV shadowing. The final amplicons were then subcloned into
the plasmid vector pCR2.1TM (Invitrogen) and sequenced on
both strands by a cycle sequencing method.
Cloning of the Rat HKg Subunit cDNA--
The
encoding DNA of the rat HK
g subunit was PCR-amplified
from rat stomach cDNA using primers flanking the coding region: sense 5
- ATAAGCTTCAGCCCTGCAGGAGAAG-3
(nucleotides +16 to
+32 of the published sequence (25)) and antisense 5
-
ATTCTAGATTACTTCTGTATTGTGAGC-3
(nucleotides +878 to +896 of
the published sequence). HindIII and XbaI sites
(underlined) were added to the 5
ends of the sense and antisense
HK
g primers, respectively, to facilitate subcloning. The
resultant amplicon was digested with HindIII and
XbaI and subcloned into these sites of the mammalian
expression vector pcDNA3.1+/Zeo (yielding the recombinant
pcDNA3.1/HK
g-Zeo). The insert HK
g DNA
was sequenced to verify its authenticity.
Primer Extension--
Antisense primers (Fig. 1A)
specific for HK2a (P7) and HK
2b (P8 and P9) were 5
-end-labeled
with [
-32P]ATP using T4 polynucleotide kinase. The
primers were annealed to 10 µg of total RNA from distal colon at
58 °C for 20 min. After cooling at room temperature for 10 min, the
primers were extended with avian myeloblastosis virus reverse
transcriptase at 42 °C for 15 min in a reaction mixture containing
50 mM Tris-HCl, pH 8.3, at 42 °C, 50 mM KCl,
10 mM MgCl2, 10 mM dithiothreitol,
1 mM each dNTP, 0.5 mM spermidine, and 2.8 mM sodium pyrophosphate. The reactions were stopped by the
addition of gel loading dye, and the samples were heated at 90 °C
for 10 min. The primer extension products were resolved by
electrophoresis on 8% acrylamide, 7 M urea polyacrylamide
gels in TBE buffer. The sizes of the primer extension products were
established by comparison with a sequence ladder generated by cycle
sequencing with the 32P-labeled primer used for each
extension reaction and the HK
2 partial genomic DNA clone (see
"Results") as template.
Analysis of Rat Genomic DNA--
The 5 end of the HK
2 gene
was analyzed using the Rat PromoterFinderTM DNA Walking Kit
(CLONTECH), which contains separate pools
("libraries") of uncloned, genomic DNA that have been predigested
with EcoRV, ScaI, DraI,
PvuII, or SspI and ligated to an oligonucleotide
anchor (adapter primer 1). A nested PCR approach was employed. In the first round, aliquots of each "library" were amplified with adapter primer 1 and HK
2 primer P1, using a program of 94 °C × 25 s, 72 °C × 4 min for 7 cycles, 94 °C × 25 s, 67 °C × 4 min for 32 cycles, and 67 °C × 4 min for 1 cycle. After analysis of an aliquot of the PCR products on a
1.2% agarose gel, the remaining PCR products were diluted 1:50 in
sterile deionized H20 and subjected to a second round of
PCR, using the nested adapter primer 2 and the nested HK
2 primer P2
(Fig. 1A) in a program of 94 °C × 25 s, 72 °C × 4 min for 7 cycles, 94 °C × 25 s,
67 °C × 4 min for 20 cycles, and 67 °C × 4 min for 1 cycle. The amplified products were separated by electrophoresis in a
0.9% agarose gel, subcloned into pCR2.1TM, and sequenced
on both strands by a cycle sequencing method.
RNA Isolation and Northern Analysis--
Total RNA was extracted
from selected tissues and renal parenchymal zones of normal and
K+-deprived rats using RNAzol B (Tel-Test). The samples
were quantitated by spectrophotometry at 260 nm. Isoform-specific
cDNAs of roughly comparable length (Fig. 1A) were
generated by PCR from the cloned HK2a and HK
2b cDNAs, using
primer pairs P3 + P4 and P5 + P6 (Fig. 1A) directed at the
unique 5
exonic sequences of the HK
2a and HK
2b isoforms,
respectively. Sequence analysis showed that these regions exhibited no
significant homology to each other or to any sequence in the GenBank
data base. A rat GAPDH cDNA (nucleotides 469-984, Ref. 26) was
also generated by PCR. For Northern analysis, the GAPDH and HK
2a-
and HK
2b-specific cDNAs were radiolabeled with 32P
by the random primer method according to the manufacturer's instructions (Prime-a-Gene, Promega, Madison, WI). Fifteen µg of
total RNA per lane were separated by size on 1% agarose, 2% formaldehyde gels and blotted to nylon membranes (Hybond N, Amersham Corp.). After UV cross-linking, the blots were visualized under UV
light, hybridized for 2 h at 68 °C in QuickHyb solution
(Stratagene) with probes specific for HK
2a, HK
2b, or GAPDH (as an
additional control for RNA quality and equality of sample loading and
transfer), and washed to a final stringency of 0.1 × SSC, 0.1%
(s/v) SDS at 60 °C. Autoradiographs of the blots were prepared at
70 °C. In several experiments (as indicated in the figure
legends), the blots were sequentially hybridized with HK
2a and
HK
2b DNA probes of comparable size and specific activity, followed
by the GAPDH DNA probe, with the blots being stripped before proceeding
to the next analysis. After each stripping, autoradiographs of the blots were prepared to verify removal of the probe.
In Vitro Transcription and
Translation--
pcDNA3.1/HK
2b-Neo and the HK
2a encoding
DNA subcloned into the vector pAGA2 (16) were transcribed and
translated in the presence of [35S]methionine with T7 RNA
polymerase and the TNT-coupled reticulocyte lysate kit (Promega,
Madison, WI). The synthesized proteins were separated by
SDS-polyacrylamide gel electrophoresis and analyzed by
fluorography.
Cell Culture and Transfection--
HEK 293 cells were grown in
modified Eagle's medium containing 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml selenium, and 2 mM
L-glutamine (complete medium). Subconfluent HEK 293 cells grown on 10-mm culture dishes were transfected with pcDNA3.1/Neo (as a vector control) or pcDNA3.1/HK
2b-Neo with the Tfx-50
reagent (Boehringer Mannheim) to yield HEK-NEO and HEK-HK
2b cell
lines, respectively. In brief, 10 µg of plasmid DNA and 22 µl of
Tfx-50 reagent were mixed with 6 ml of modified Eagle's medium. The
mixture was added to the monolayers and incubated for 2 h at
37 °C in a 5% CO2 incubator. Twelve ml of prewarmed
complete medium was then overlaid onto the medium, and the cells were
returned to the incubator. After 48 h, the medium was replaced
with complete medium containing 600 µg/ml G418 (Life Technologies,
Inc.). The G418-containing medium was replaced every 3 days until
individual resistant colonies were isolated and established in culture
as individual lines. All lines were maintained in G418 medium and frozen after one to three in vitro passages. HEK-HK
2b
clone 25 was used in the functional analysis detailed below. To test
whether coexpression of the HK
g affected functional
expression of HK
2b, HEK-NEO and HEK-HK
2b cell lines were stably
transfected with pcDNA3.1/HK
g-Zeo and selected in
complete medium containing 600 µg/ml G418 and 250 µg/ml Zeocin.
Cells surviving selection were screened for HK
2b and/or
HK
g expression by Northern analysis with probes specific
for each subunit. The doubly transfected cells were termed
HEK-HK
2b/HK
g, and clone 40 was selected for further
functional analysis.
86Rb+ Uptake--
Uptake of
86Rb+, a K+ congener, was measured
at 37 °C in transfected HEK 293 cells grown in 24-well plates
according to a published protocol (22). Monolayers were rinsed five
times and preincubated in uptake buffer (145 mM NaCl, 1 mM KCl, 10 mM glucose, 1.2 mM MgCl2, 1 mM CaCl2, 2 mM
NaH2PO4, 32 mM HEPES, pH 7.4, and
200 µM bumetanide) at 37 °C for 20 min in the presence
or absence of different concentrations of ouabain as indicated in the
figure legends. External 1 mM K+ was used in
these assays, because K+ competitively inhibits both Sch
28080 and ouabain binding to X+-K+-ATPase subunits, and this
concentration is within the narrow range of Km
values reported for K+ dependence of all known
X+-K+-ATPase
subunits. Uptake
was initiated by adding 0.2 ml of uptake buffer containing ~4
µCi/ml 86Rb+. After 12 min, the reaction was
stopped by six rapid washes with ice-cold stop buffer (100 mM MgCl2, 10 mM Tris-HEPES, pH
7.4). Parametric studies indicated that this time point was in the
linear range of uptake. The cells were solubilized in 2% SDS, 0.1 N NaOH, and the resulting extracts were measured for
86Rb+ by Cerenkov radiation and for protein
content by the BCA Protein Assay Reagent (Pierce). Triplicate or
quadruplicate measurements were obtained in each uptake condition.
Data Analysis--
The intensities of bands on the Northern blot
autoradiograms were measured by whole band densitometry software
running on a SPARC Station IPC (Sun Microsystems, Mountain View, CA)
equipped with an image analysis system (BioImage, Ann Arbor, MI).
Predictions of membrane-spanning regions and their orientation were
generated by the TMpred program (27) through the ISREC Bioinformatics Group server. Predictions of potential promoter regions were obtained with a neural networks algorithm (28) through the LBNL Human Genome
Informatics Group server. Potential regulatory motifs in the HK2
gene were identified with Transcription Element Search Software from
the Computational Biology and Informatics Laboratory server of the
University of Pennsylvania School of Medicine, using the Transfac 3.1 data base. Quantitative data are presented as mean ± S.E. and
were analyzed for significance by analysis of variance. Significance
was assigned at p < 0.05.
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RESULTS |
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cDNA Cloning and Structural Analysis of a Truncated N-terminal
Variant of the H+-K+-ATPase 2
Subunit--
The anchor-ligated cDNAs synthesized from rat kidney
mRNA were subjected to 5
-RACE using adapter primer 2 and
HK
2-specific primer P1 from exon 2 (Fig.
1A). Two distinct PCR products
of ~400 and ~600 bp, subsequently shown to correspond to the 5
ends of HK
2a and HK
2b, respectively, were consistently amplified. These products were isolated, subcloned, and sequenced. A total of 16 RACE reactions for both amplicons was analyzed in this manner. The two
RACE product subtypes differed in sequence at their 5
ends but were
identical at their 3
ends, with common sequence beginning at the codon
for Lys4 of the known HK
2a sequence (Fig.
1A). HK
2a was identical in sequence to the corresponding
region of the HK
2 cDNA reported by Crowson and Shull (17) but
included an additional 72 bp at its 5
end, so that the total 5
-UTR
was 274 bp.
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Genomic Organization of the 5 End of the HK
2 Gene and Mapping
of the Transcription Start Sites--
Analysis of the HK
2a and
HK
2b 5
-RACE products suggested that both mRNAs are derived from
a single gene by utilization of alternative splice sites at the 5
region. To determine the order of the exons and the intervening genomic
sequences, we used nested reverse primers derived from the 5
region
common to both variants and adapter-ligated rat genomic DNA libraries
to PCR amplify a portion of the 5
region of the HK
2 gene. PCR
products of ~1.6- and 0.5-kb were consistently amplified from the
DraI and PvuII libraries, respectively. These
amplicons were subcloned into pCR2.1TM and sequenced on
both strands. Sequence analysis indicated that the HK
2b transcript
is the product of an alternative transcription initiation site located
within the first intron (Fig. 1, A and B). The 5
end of the HK
2b mRNA represents a 5
extension of exon 2 that is
excised in the HK
2a mRNA. In support of this construct, a
consensus 5
splice donor site (5
-GTGAGT-3
) was identified at the
exon 1/intron 1 boundary, and a consensus 3
acceptor site (CAG) (29)
was found in the expected 5
region of exon 2 (Fig. 1A).
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Analysis of Potential Gene Control Elements--
The HK2
partial genomic clone contained sequences of ~380 and ~205 bp
immediately 5
to the transcription start sites of the HK
2a and
HK
2b transcription units, respectively. These sequences share no
obvious homology, and they were examined for potential DNA elements
that may contribute to transcriptional initiation and regulation. The
HK
2a 5
-flanking region contains a TATA-like sequence (ATTTAA), a
CACCC sequence (31), and a CCAAT motif (30) beginning 27, 127, and 133 bp, respectively, 5
to the transcription start site, which likely
comprise the core promoter module (Fig. 1A). The 380 bp
immediately preceding the transcription start sites contains several
potential cis-elements that may serve as binding sites for
transcription factors. These include 7 Sp 1 sites (32), 3 AP-2 sites
(33), 2 GR sites (31), and single GATA-1 (34), C/EBP (35), PEA-3 (36),
NF-
B (37), and HNF-4 (38) motifs (Fig. 1A).
HK2 Isoform mRNAs Are Expressed in Colon and
Kidney--
Northern blots of total RNA harvested from an array of
tissues harvested from
K+-replete rats were probed with 32P-labeled
DNA probes specific for each HK
2 subtype (Figs. 5 and 6). Both isoforms were expressed
prominently in the distal colon (Fig. 5) and very weakly in the
proximal colon and normal kidney (Fig. 6). No transcripts were detected
in skeletal muscle, heart, brain, stomach, spleen, liver, testis, or
lung (Fig. 5), even with prolonged autoradiographic exposures. Failure
to detect transcripts in these latter tissues also indicates that the
HK
2 isoform-specific probes did not cross-hybridize with the four
known Na+-K+-ATPase
subunit isoforms
(abundantly expressed in heart, brain, skeletal muscle, and/or testis
(41, 42)) or the HK
1 subunit (abundantly expressed in stomach (14)).
Moreover, reprobing the blots with a 32P-labeled DNA probe
for GAPDH indicated comparable abundance and integrity of the blotted
RNA samples (data not shown).
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Expression of HK2 Subunit Isoform mRNAs in Kidney and Colon
of Control and K+-restricted Rats--
To determine the
response of the HK
2a and HK
2b gene products to dietary
K+ restriction in rat kidney and colon, Northern analysis
with 32P-labeled DNA probes specific for each HK
2
subtype was performed on total RNA harvested from the proximal colon,
distal colon, and renal cortex, outer medulla, and inner medulla of
control and K+-restricted rats. In control rats, an
abundant ~4.0-kb subunit transcript (HK
2b
HK
2a) was
detected with both the HK
2a- and HK
2b-specific probes in distal
colon (Fig. 6A). In addition, the HK
2b-specific probe
hybridized to a much less abundant ~6.0-kb transcript in distal colon
(Fig. 6A). It is not known whether this larger transcript
represents a processing intermediate or an mRNA with an alternate
polyadenylation signal, but similar results were reported by Crowson
and Shull (17), who used C-terminal coding and 3
-UTR sequences as
probes. With prolonged autoradiographic exposures (3 days), very low,
comparable levels of HK
2a subunit mRNA were detected in the
proximal colon, cortex, and outer and inner medulla (data not shown).
When these blots were reprobed with the HK
2b-specific probe of
roughly comparable size and specific activity, detectable ~4.0-kb
transcripts were observed in the same structures after overnight
exposure, suggesting that HK
2b is expressed at higher levels, albeit
still very low, than HK
2a in normal kidney and colon.
Functional Expression of the H+-K+-ATPase
2b Subunit in HEK 293 Cells--
A dual selection strategy, using
separate mammalian expression vectors containing the encoding DNAs for
HK
2b and HK
g together with the neomycin and Zeocin
resistance genes, respectively, was employed to generate cell lines
stably expressing the HK
2b subunit, the HK
g subunit,
or both subunits. HEK 293 cells were chosen as the recipient cells for
the transfection experiments because they are easily transfected, do
not express H+-K+-ATPase
or
subunit
gene products, their endogenous Na+-K+-ATPase
is highly sensitive to ouabain (22), and they permit analysis of
H+-K+-ATPase biosynthesis and subunit assembly
in mammalian cells at 37 °C (a factor that has been suggested to
influence the fidelity of oligomerization and membrane insertion of the
pump, Ref. 22).
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DISCUSSION |
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Analysis of the regulation of active K+ reabsorption
in the renal collecting duct and distal colon has been hampered by the lack of structural data concerning potential control mechanisms governing HK2 gene expression. In this study, we characterized two
alternatively spliced products of the rat HK
2 gene, HK
2a and
HK
2b, that apparently arise from the use of alternative promoters and differ in the length of their N termini and their relative abundance in kidney and colon. Heterologous expression studies of the
novel transcript in HEK 293 cells indicate that HK
2b encodes a
plasma membrane mechanism for K+ uptake that, like that of
the full-length HK
2 subunit (16, 18, 19), is relatively Sch
28080-resistant, intermediate in its sensitivity to ouabain, and
operates more effectively when coexpressed with the HK
g
subunit. The HK
2b isoform represents the most abundantly expressed
HK
2 transcript in the rat kidney and distal colon and the principal
H+-K+-ATPase transcript up-regulated in the
renal medulla of K+-deprived rats. We also identified
structural features that may govern transcriptional initiation and
control as well as translational regulation of these isoforms. Our
results suggest that both HK
2 isoforms may contribute to
K+ conservation during chronic hypokalemia, and they
uncover a new degree of regulatory complexity for the
X+-K+-ATPase
subunit gene
family.
The first variant, HK2a, is the previously described (17) 1036-amino
acid protein, which is encoded by a 4.0-kb mRNA transcribed from
the 5
-most putative promoter. Primer extension analysis places the
major transcription initiation site 274 bp upstream of the translation
initiation methionine. Exon 1 includes the 5
-UTR and encodes the first
three amino acids of the primary HK
2a translation product. This
structural theme is common to other members of the
X+-K+-ATPase
subunit family. The
analogous exon in ATP1AL1 also encodes 3 amino acids,
whereas those for the HK
1, and Na+-K+-ATPase
1 and
2 subunits genes encode 4 amino acids, and that for the
Na+-K+-ATPase
3 subunit gene encodes only 2 amino acids. The 5
-flanking region of HK
2a contains common basal
promoter elements. An AT-rich sequence that might serve as a TATA
element begins 21 bp 5
to the transcription start site. This sequence
is preceded by potential CCAAT (30) and CACCC (31) elements residing
within the preferred context for such elements. In addition, sequence
inspection of the 5
-flanking regions revealed potential
cis-acting DNA elements, including sites for AP-2, AP-3,
GATA-1, HNF-4, C/EBP, GR, PEA-3, NF-
B, and multiple Sp1 sites
sequences that may participate in transcriptional regulation of this
gene. Of these, Sp1 (43) and GATA DNA-binding proteins (44) have been
shown to play important roles in transcriptional activation of the
HK
1 gene. Since we did not confirm the 5
end of the HK
2 gene,
other potential regulatory elements may reside upstream of the sequence
we characterized.
The second variant, HK2b, has not been previously recognized. This
929-amino acid protein is also encoded by an ~4.0-kb mRNA that is
transcribed from an internal putative promoter residing in intron 1. Given the near-identical size of the major HK
2a and HK
2b mRNA
transcripts, Northern analysis with probes directed to sites distal to
the alternative splice site in exon 2 would be unable to distinguish
between the two isoforms. Our DNA sequence data (Fig. 1A)
combined with the in vitro transcription and translation results (Fig. 3) and functional expression data (Fig. 7, B
and C) indicate that the HK
2b isoform encodes a protein
with the requisite features of an
X+-K+-ATPase
subunit. Primer
extension and 5
-RACE identified a putative site for HK
2b
transcription initiation, but given the context of the surrounding
nucleotides, additional transcription start sites may be located
upstream of the site we identified (that is closer to the TATA, CACCC,
and reverse complement CCAAT sequences found in the 5
-flanking region
of the HK
2b transcription unit). Other potential
cis-elements, including multiple AP-2 and Sp-1 sites, as
well as consensus NF-interleukin 6, IRF-1, AP-4, GR, and GATA-1
sequences, were identified in this region. Conclusive evidence for the
functional activity of the HK
2a and HK
2b promoter elements will
require formal testing with promoter-reporter gene constructs.
A notable feature of the HK2b mRNA is the complex 5
-UTR
containing multiple, partially overlapping AUG triplets in ORFs upstream (uORF) of the translation initiation site of the major ORF.
Recent analyses have shown that such uORFs are present in <10% of
vertebrate mRNAs (45) and that in some instances they inhibit
translational initiation at the major ORF. For example uORFs in the
5
-UTR of the retinoic acid receptor
2 and transforming growth
factor
3 mRNAs dramatically inhibited CAP-dependent
translation in vitro (46, 47). Moreover, studies of the
retinoic acid receptor
2 mRNA in transgenic mice indicate a role
for uORFs in tissue-specific and developmentally regulated gene
expression (48). The relatively low level of
86Rb+ uptake activity attributable to the
HK
2b pump in the HEK-HK
2b and HEK-HK
2b/HK
g cell
lines (Fig. 8A) despite abundant mRNA expression (Fig.
7A) might reflect this regulatory constraint. Alternatively,
preference of HK
2b for an
X+-K+-ATPase
subunit other than
the endogenous Na+-K+-ATPase
1 subunit
expressed in HEK 293 cells might limit expression of transport
activity. The fact that coexpression of the HK
g subunit
supported higher rates of 86Rb+ uptake activity
and was required for survival in 1 µM ouabain supports
this latter hypothesis. Since studies of the full-length HK
2
subunit, expressed by cRNA injection in Xenopus oocytes, indicated that the rat Na+-K+-ATPase
1 and
HK
g subunit support comparable rates of K+
uptake (16), it remains to be determined whether the two HK
2 isoforms differ in their promiscuity for
X+-K+-ATPase
subunits.
The functional and regulatory significance of the N-terminal truncation
of HK2b remains to be explored in further detail. The N terminus is
the most variable structural region among the X+-K+-ATPase
subunits.
Conceivably the decision to code for the N-terminal 108 amino acids
present in HK
2a could dictate isoform-specific differences in
membrane targeting or cytoskeletal association in polarized epithelia,
regulation by protein kinase C or protein kinase A phosphorylation
(since these sites are present in HK
2a but not HK
2b), ion
transport kinetics, or inhibitor sensitivities. There is precedent for
alternative promoters to direct the coding of protein variants that are
targeted to different intracellular locales. The two variants of
leukemia inhibitory factor, which exist as diffusible and extracellular
matrix-associated isoforms, represent such an occurrence (49). The
possibility for functional and pharmacological differences in the
HK
2 isoforms is particularly intriguing since N-terminal deletion
mutants of the closely related Na+-K+-ATPase
exhibited altered K+ deocclusion kinetics compared with
wild-type pumps (50), and since the N-terminal truncation of HK
2b
impinges on the H1-H2 domains, which have been implicated in ouabain
and Sch 28080 binding to other
X+-K+-ATPase
subunits (reviewed
in Ref. 13). However, like the full-length HK
2 subunit (16, 18, 19),
HK
2b is insensitive to high concentrations of Sch 28080. Moreover,
the approximate IC50 for ouabain inhibition (400-800
µM in the presence of external 1 mM
K+) of the HK
2b/HK
g pump reported here is
comparable to values reported for the full-length HK
2 subunit
expressed in heterologous systems. Codina et al. (16)
reported an IC50 of 400-600 µM in the
presence of external 1 mM K+ for HK
2 pumps
expressed in Xenopus oocytes, and Cougnon et al. (18) reported Ki values for ouabain of ~70 and
~970 µM in the presence of external 0.2 and 5 mM K+, respectively, for HK
2 pumps expressed
in HEK 293 cells. Clearly, heterologous expression and detailed
functional analysis of the two isoforms in a common host cell will be
needed to distinguish subtle differences.
These considerations take on added meaning when viewed in the context
of recent functional studies in kidney and colon. In vitro
studies have identified at least three different K+-ATPase
activities that are distinguished by their kinetic and pharmacological
properties in rat kidney (51, 52). One activity (type I) is
K+-, but not Na+-dependent,
ouabain-resistant, Sch 28080-sensitive, and expressed in collecting
ducts. A second activity (type II) is K+-, but not
Na+-dependent, Sch 28080- and
ouabain-sensitive, and expressed basally in proximal tubules and the
thick ascending limbs (52). This activity is virtually abolished during
chronic K+ depletion. A third activity (type III) is
activated by either Na+ or K+, exhibits higher
sensitivities to ouabain and to Sch 28080 than type II, and a lower
sensitivity to Sch 28080 than type I. This activity is not expressed
basally but is specifically up-regulated in cortical collecting
ducts and OMCDs with chronic hypokalemia (52). Similarly, both
ouabain-sensitive and insensitive K+-ATPase
activities have been identified in the apical membranes of colonocytes
from the distal colon (10), yet only HK2 mRNA (5, 8) and protein
(8) have been identified in these cells. These collective data have led
us to postulate that a yet-to-be discovered K+-ATPase
isoform may be operative in the renal collecting duct and colon (8, 19,
51). It is possible that functional differences in the HK
2 protein
variants may account for these puzzling data.
In addition to the generation of protein isoforms differing at the N
terminus, the use of alternative promoters in the HK2 gene would be
expected to afford considerable versatility in controlling its
expression. Alternative promoter usage in other genes has been shown to
allow for expression of isoforms exhibiting differences in the degree
and timing of transcription initiation, mRNA turnover, translational efficiency, tissue specificity, and responses to signal
transduction pathways (53). The HK
2 gene appears to be the first
example of a P-type ATPase to employ alternative promoters and mRNA
splicing to generate structural and regulatory diversity. This
mechanism, then, adds to the known complexity of
X+-K+-ATPase regulation, which
includes controls on transcription, translational efficiency, subunit
assembly, and various post-translational modifications. It may also
provide an explanation for the well documented differential expression
of the HK
2 gene in kidney and distal colon under various
experimental conditions. For example, Jaisser and co-workers (5) showed
that chronic K+ deprivation did not alter, adrenalectomy
reduced, and dexamethasone supplementation of adrenalectomized
rats restored steady-state HK
2 mRNA levels in distal colon. In
contrast, chronic K+ deprivation enhanced HK
2 mRNA
expression in the OMCD, whereas adrenalectomy did not alter HK
2 gene
expression. We (6) and others (7) have shown similar effects of
K+ deprivation on HK
2 mRNA levels in the OMCD. The
probes used in all these studies would be expected to hybridize to both
HK
2 variants. Similarly, Sangan and colleagues (8) showed that chronic dietary Na+ depletion (presumed to promote
secondary hyperaldosteronism), but not chronic K+
depletion, enhanced HK
2 mRNA and protein levels in distal colon. Conversely, chronic K+ depletion promoted HK
2 protein
but not mRNA expression in outer medulla, whereas Na+
depletion did not affect renal expression of this gene product. Fortuitously, the antibody (termed M-1) used in this and an earlier (19) study was raised against a fusion protein produced from the first
109 amino acids of the HK
2a sequence. Thus, this antibody would be
expected to be specific for HK
2a, and it would not detect HK
2b.
M-1 immunoreactivity was identified in the apical membranes of
principal cells of the K+-deprived OMCD (8) and of surface
cells in rat distal colon (19). Since the consensus of in
situ hybridization studies (5, 6) with probes common to the two
HK
2 variants indicated that HK
2 mRNA is primarily expressed
in OMCD intercalated cells, it is reasonable to hypothesize that the
two isoforms are expressed in different cell types of the rat OMCD
during K+ depletion. The sequence information presented
here should facilitate future studies to define the molecular
mechanisms controlling the differential and cell type-specific
expression of these isoforms.
Finally, although it has been hypothesized that HK2 and ATP1AL1
represent species variants of the same protein, the novel structural
organization and regulatory mechanisms for HK
2 transcription described here add to the growing list of differences that suggest that
these proteins represent distinct protein isoforms. These distinguishing features include the contrasting tissue distributions (20), differences in pharmacological profile (16, 18, 19, 21, 22), and
greater degree of sequence divergence when compared with the
interspecies differences of the other human and rat
X+-K+-ATPase
subunit isoforms.
As additional structure-function and structure-regulation correlations
for these genes are identified, their evolutionary relationship should
come into clearer focus.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Gary Shull (University of
Cincinnati) for the generous gift of the
H+-K+-ATPase 2 cDNA and Dr. Juan Codina
(University of Texas-Houston) for the gift of the
H+-K+-ATPase
2 cDNA subcloned in
pAGA2.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U94911-U94913.
Supported by National Institutes of Health Grants 1R29 DK47981 and
1R01 DK50745 and an Established Investigatorship from the American
Heart Association. To whom correspondence should be addressed: Depts.
of Internal Medicine and of Integrative Biology, Pharmacology and
Physiology, The University of Texas Medical School, 6431 Fannin, MSB
4.148, Houston, TX 77030. Tel.: 713-500-6870; Fax: 713-500-6890 or
6882; E-mail: bkone{at}heart.med.uth.tmc.edu.
1
The abbreviations used are: HK2,
H+-K+-ATPase
2 subunit, also termed the
colonic H+-K+-ATPase
subunit; HK
1,
H+-K+-ATPase
1 subunit, also termed the
gastric H+-K+-ATPase
subunit;
HK
g, gastric H+-K+-ATPase
subunit; OMCD, outer medullary collecting duct; RACE, rapid
amplification of cDNA ends; C/EBP, CCAAT enhancer binding protein;
NF-
B, nuclear factor kappa B; HNF-4, hepatocyte nuclear factor-4;
IRF-1, interferon regulatory factor-1; GR, glucocorticoid receptor;
UTR, untranslated region; u, upstream; ORF, open reading frame; Sch
28080, 2-methyl,8-(phenylmethoxy)imidazo(1,2-a)pyridine 3-acetonitrile; kb, kilobase pair(s); bp, base pair(s); PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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REFERENCES |
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