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J Am Soc Nephrol 12:2554-2564, 2001
© 2001 American Society of Nephrology

Structure, Promoter Analysis, and Chromosomal Localization of the Murine H+/K+-ATPase {alpha}2 Subunit Gene

Wenzheng Zhang*, Teresa Kuncewicz*, Sandra C. Higham* and Bruce C. Kone*{dagger}

Departments of *Internal Medicine and {dagger}Integrative Biology, Pharmacology, and Physiology, The University of Texas Medical School at Houston, Houston, Texas.

Correspondence to Dr. Bruce C. Kone, Departments of Internal Medicine and Integrative Biology, Pharmacology, and Physiology, The University of Texas Medical School at Houston, 6431 Fannin, MSB 4.138, Houston, TX 77030. Phone: 713-500-6870; Fax: 713-500-6890 (or 6882); E-mail: Bruce.C.Kone{at}uth.tmc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. The H+/K+-ATPase {alpha}2 subunit (HK{alpha}2) of distal colon and renal collecting ducts plays a critical role in potassium and acid-base homeostasis. The isolation and complete sequence of the murine HK{alpha}2 gene are reported. The HK{alpha}2 gene contains 23 exons and spans 23.5 kb of genomic DNA. The exon/intron organization is comparable to that of the human ATP1AL1 gene. Primer extension and 5'-rapid amplification of cDNA ends of distal colon RNA were used to map the transcription initiation site. Fluorescence in situ hybridization analysis localized the HK{alpha}2 gene to murine chromosome 14C3. Sequence analysis of 7.2 kb of the 5'-flanking region revealed numerous consensus sites for transcription factors, including two potential glucocorticoid response elements. Transient transfection of promoter-luciferase constructs demonstrated strong basal HK{alpha}2 promoter activity in renal collecting duct cells but not in fibroblasts or in a medullary thick ascending limb of Henle’s loop cell line. Deletion analysis revealed that the proximal 0.2 kb of the promoter was sufficient to confer activity in collecting duct cells. These data should prove important in elucidation of the mechanisms controlling the differential, tissue-specific expression of the HK{alpha}2 gene.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Maintenance or restoration of the K+, Na+, and acid-base balance is critical for the normal physiologic processes of all eukaryotic cells. Perturbations in the normal or adaptive mechanisms controlling this homeostasis can rapidly lead to organ system dysfunction and death. Consequently, the body has evolved a complex integrated system, principally involving epithelial cells of the kidney and colon, to adjust K+, Na+, and acid elimination in response to dietary intake. A concordance of functional, molecular biologic, and cell biologic data from experimental animals suggest that members of the H+/K+-ATPase gene family participate importantly in the control of body K+ balance (1,2). Indeed, mice with targeted ablation of the gene for H+/K+-ATPase {alpha}2 subunit (HK{alpha}2) (also termed the "colonic" H+/K+-ATPase) develop profound hypokalemia, principally because of fecal K+ wasting (3). HK{alpha}2 may also contribute to bicarbonate absorption by the kidney (4) and distal colon (5) and enhanced ammonium secretion in the inner medullary collecting duct (IMCD) during chronic hypokalemia (6). Emerging data also suggest a role for HK{alpha}2 in chronic adaptation to changes in sodium (7) and aldosterone (8,9) balance, perhaps operating in a Na+/K+ exchange mode (7).

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 mechanisms and a requirement for a heterodimeric ({alpha}/ß) structure. The X+/K+-ATPase {alpha} subunits share considerable (approximately 65%) amino acid homology and contribute most of the functional properties of the holoenzymes, but they can be distinguished on the basis of their organ distributions and sensitivities to the inhibitors ouabain and Sch 28080 [2-methyl,8-(phenylmethoxy)imidazo(1,2-{alpha})pyridine-3-acetonitrile] (1). The H+/K+-ATPase {alpha}1 subunit was first cloned from and is principally expressed in stomach (10), where it participates in gastric acid secretion. The H+/K+-ATPase {alpha}1 subunit protein is highly sensitive to inhibition by Sch 28080 and is resistant to ouabain inhibition (11). The HK{alpha}2 cDNA was first cloned from rat distal colon (12), where it is abundantly expressed; lower levels of HK{alpha}2 mRNA were observed in proximal colon (12), uterus (12), and kidney (8,13). Orthologs of this cDNA have since been cloned from guinea pig (14), rabbit (15), and human (ATP1AL1) (16) sources. These orthologs share approximately 84% amino acid sequence identity across species. Alternative promoter usage and transcript splicing lend structural diversity to HK{alpha}2 in rats (17) and rabbits (15), but the physiologic roles of the resulting proteins are unclear.

The human ATP1AL1 gene was originally identified during genomic library screening for Na+/K+-ATPase gene family members (18). The ATP1AL1-encoding DNA was subsequently isolated from a human skin cell library (16). ATP1AL1 transcripts are basally expressed principally in skin, kidney, and brain (16,19,20), which is a distribution pattern somewhat different from that observed in other species. The human ATP1AL1 gene contains 23 exons and spans approximately 32 kb of genomic DNA on human chromosome 13 (19). A limited, 1.4-kb stretch of the proximal 5'-flanking region of the ATP1AL1 gene was also characterized. The ATP1AL1 locus was first mapped to 13q21–q31 by Southern analysis of DNA from panels of rodent/human somatic cell hybrids (18). In subsequent linkage map analyses, the ATP1AL1 locus was placed much more proximally, at 13q12.1–q12.3 (21,22). Given the differences in the tissue distribution and pharmacologic profile of ATP1AL1, compared with HK{alpha}2 in other species, there has been some controversy regarding whether ATP1AL1 represents an ortholog of HK{alpha}2 or is indeed a novel X+/K+-ATPase. Moreover, no study has examined the mechanisms governing HK{alpha}2 transcription.

In this study, we isolated and completely sequenced the cDNA and structural gene encoding the murine HK{alpha}2 gene, determined its transcriptional start site, and defined its chromosomal location. We also sequenced 7.2 kb of the proximal 5'-flanking region of the gene and demonstrated the functional activity of this promoter in cultured renal collecting duct cells. In addition, we have isolated a bacterial artificial chromosome (BAC) clone containing the murine HK{alpha}2 gene, which should prove useful in defining regulatory elements that control transcription of this gene.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Oligonucleotide Synthesis and DNA Sequence Analysis
Genosys (The Woodlands, TX) synthesized all oligonucleotides. All cDNA and genomic sequencing was performed at the Molecular Genetics Core Facility of the University of Texas Medical School at Houston, by using double-stranded templates and a model 377A automated DNA sequencer (Applied Biosystems, Foster City, CA), according to the standard protocol for the Taq DyeDeoxy terminator cycle sequencing kit (Applied Biosystems).

Isolation and Sequence Analysis of {lambda} Phage Clones Comprising the Murine HK{alpha}2 Gene
A 129/SvJ, spleen-derived, mouse genomic library in the Lambda Fix II vector was obtained from Stratagene (La Jolla, CA). Approximately 500,000 recombinants were plated, and duplicate filters were screened with a 32P-labeled, 808-bp, murine HK{alpha}2a genomic fragment [corresponding to the 5'-untranslated region (UTR) in exon 1, the first intron, and the first portion of exon 2 that we had PCR-cloned from C57BL/6J mouse tail genomic DNA]. Filters were prehybridized and hybridized for 16 h at 45°C in ULTRAhyb solution (Ambion, Austin, TX). Filters were washed at 45°C in 2x SSC/0.1% sodium dodecyl sulfate (SDS), followed by 0.1x SSC/0.1% SDS, and were then exposed to autoradiographic film overnight at -70°C, with intensifying screens. Multiple positive clones were identified, and one (clone {lambda}63a-1) was plaque-purified by two additional rounds of screening. The library was rescreened with a cDNA probe comprising nucleotides +1658 to +3328 of the rat HK{alpha}2 cDNA. Three additional clones, namely {lambda}4a-1, {lambda}4b-1, and {lambda}5a-2, were identified. Phage DNA from the aforementioned clones was purified and subjected to restriction enzyme digestion and sequence analysis. To accelerate the sequencing of these phage clones, primers were designed according to the sequences that were highly conserved between rat HK{alpha}2a and human ATP1AL1 mRNA and within an exon, according to human ATP1AL1 exon/intron boundaries. These primers were used either to directly sequence the phage DNA or to amplify PCR products from these phage clones. PCR products were then sequenced.

Cloning of a BAC Containing the Murine HK{alpha}2 Gene
The RPCI-23 BAC library (23), constructed from the C57BL6/J mouse strain, was used for isolation of the murine HK{alpha}2 gene. The library had been gridded as a 4 x 4 array, with internal duplicates, onto 22- x 22-cm, nylon, high-density filters for screening by probe hybridization. Each hybridization membrane represented 18,432 independent mouse BAC clones, stamped in duplicate. The average insert size is 197 kb and represents an 11-fold genome representation. The high-density BAC filters were hybridized with [32P]dATP- and [32P]dCTP-labeled probes generated from overlapping oligonucleotides (overgos), according to the method described by McPherson (24). The three probes represented nucleotides +101 to +136 in the 5'-UTR, +1955 to +1990 in the middle of the coding region, and +2932 to +2966 at the end of the coding region of the rat HK{alpha}2 cDNA (12). Subsequent analysis of the murine HK{alpha}2 cDNA revealed that the rat overgo probes were 85, 92, and 95%, respectively, identical to the murine HK{alpha}2 cDNA. Filters were prehybridized for 4 h at 58°C in hybridization solution 1 (24), containing 0.5 M sodium phosphate, 1 mM ethylenediaminetetraacetate, 7% SDS, and 1% bovine serum albumin, and were then hybridized overnight at 58°C with the probe in fresh hybridization solution 1. The filters were then successively washed in 2x SSC at room temperature, 2x SSC at 50°C, and 1.5x SSC at 50°C. The filters were exposed to XAR5 film (Eastman Kodak, Rochester, NY) at -70°C. Ten clones that hybridized to all three overgo probes were identified. Clone RP23-442H19 was subjected to partial sequence analysis, using primers from the 5'- and 3'-ends of the murine HK{alpha}2 gene, to confirm its molecular authenticity.

5'- and 3'-Rapid Amplification of cDNA Ends to Clone Murine HK{alpha}2 cDNA
The 5'-rapid amplification of cDNA ends (RACE) protocol was performed using the Marathon cDNA amplification kit (Clontech, Palo Alto, CA), according to the instructions provided by the manufacturer. First-strand cDNA were generated from 1 µg of mouse colon RNA, using Moloney murine leukemia virus reverse transcriptase and a modified locking oligo(dT) primer (containing two degenerate nucleotide positions at its 3'-end) that was provided with the kit. Second-strand synthesis was accomplished with a cocktail 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) by using T4 DNA ligase. The anchor-ligated cDNA were then subjected to 5'-RACE using a nested primer (adapter primer 2, supplied with the RACE kit) complementary to adapter primer 1, the HK{alpha}2-specific reverse primer 5'-CTCTGGAGTTTGCTTGGGAG-3', which is complementary to nucleotides +284 to +303 of the published rat HK{alpha}2 cDNA sequence (12), and the components of the Advantage cDNA amplification kit (Clontech). PCR cycling conditions were 94°C for 1 min, followed by 28 cycles of 94°C for 30 s and 68°C for 4 min, with a final step of 68°C for 4 min. Ten microliters of the amplified products were separated by electrophoresis in a 1% agarose gel and were observed by ethidium bromide staining and ultraviolet shadowing. The final amplicons were then subcloned into the plasmid vector pCR2.1 (Invitrogen, San Diego, CA) and sequenced. 3'-RACE was performed by using 3'-RACE-ready cDNA and the SMART RACE cDNA amplification kit (Clontech), according to the protocol described by the manufacturer. The murine HK{alpha}2 cDNA-specific forward primer 5'-ATGAGACGGTGGAAGACATCGCAAAACG-3' (+1946 to +1973) was used in the PCR. Amplification products were analyzed, subcloned, and sequenced. After the 5'- and 3'-ends of the cDNA had been determined by these methods and comparison with the genomic sequence information, the full-length cDNA was PCR-amplified from mouse colon cDNA by using primers directed to the 5'-end, as determined by 5'-RACE and primer extension, and the 3'-end, as identified from the genomic clone and 3'-RACE results.

Primer Extension Assay
The primer extension assay was performed according to methods previously established in our laboratory (17). Antisense primer specific for the murine HK{alpha}2 cDNA (5'-TTGCCTTAGCGGCGCGCTGGACTG-3', antisense for nucleotides +139 to +162) was 5'-end-labeled with [{gamma}-32P]ATP, using T4 polynucleotide kinase. The primers were annealed to 10 µg of total RNA from distal colon for 20 min at 58°C. After cooling at room temperature for 10 min, the primers were extended with avian myeloblastosis virus reverse transcriptase for 15 min at 42°C, 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. As a negative control, 50 µg of yeast tRNA was incubated with the radiolabeled oligonucleotide under the same hybridization conditions as used for the experimental samples. 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 Tris-borate-ethylenediaminetetraacetate buffer. The sizes of the primer extension products were established by comparison with 32P-labeled molecular weight standards and with a sequence ladder generated by cycle sequencing with the 32P-labeled primer used for each extension reaction, with a HK{alpha}2 partial genomic DNA clone as template.

Northern Analysis
Total RNA was obtained from mouse distal colon, ileum, kidney, uterus, brain, liver, and spleen, and Northern blots were prepared as detailed previously (17). The blots were hybridized with a 32P-labeled cDNA probe corresponding to nucleotides +1 to +272 of the murine HK{alpha}2 cDNA. Equal loading of samples on the gel and transfer to the membrane were assessed by ethidium bromide staining of 28S and 18S RNA on the gels and blots.

Transient Transfection and Reporter Gene Assays
Various segments of exon 1 (beginning at nucleotide +253, which is 3' to the transcription start site and just 5' to the translation initiation codon ATG) and the 5'-flanking region of the murine HK{alpha}2 gene were PCR-amplified from {lambda}63a-1, a phage clone identified to contain the entire 5'-portion of the murine HK{alpha}2 gene. The PCR fragments included nucleotides +253 to -177, +253 to -477, +253 to -1329, +253 to -2833, +253 to -4306, +253 to -5667, and +253 to -7265. The fragments were then subcloned into the MluI and BglII sites of pGL3-Basic, to generate the constructs pGL3-0.18MHK{alpha}2, pGL3-0.48MHK{alpha}2, pGL3-1.3MHK{alpha}2, pGL3-2.8MHK{alpha}2, pGL3-4.3MHK{alpha}2, pGL3-5.9MHK{alpha}2, and pGL3-7.2MHK{alpha}2, respectively. Four cell lines were used as recipients for the transfection experiments, i.e., mIMCD3 (25) and mOMCD1 (26) cell lines, which are derived from mouse IMCD and outer medullary collecting duct (OMCD), respectively, and are known to express HK{alpha}2 mRNA; ST-1 cells (27), which are derived from the medullary thick ascending limb of Henle’s loop (MTAL) of SV40 transgenic mice and do not express HK{alpha}2; and NIH 3T3 fibroblasts, which do not express HK{alpha}2. Each of the cell lines was grown in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, in 24-well plates. The cells were transiently transfected with a total of 1 µg/well of plasmid DNA by using Lipofectamine 2000 reagent (Life Technologies, Gaithersburg, MD), according to the protocol provided by the manufacturer. The parent vector pGL3-Basic (which lacks eukaryotic promoter and enhancer sequences) or the HK{alpha}2 promoter-luciferase constructs were added at 0.9 µg/well. For comparative purposes, the cells were cotransfected with the Renilla luciferase expression plasmid pRL-SV40 (0.1 µg/well), to control for variations in transfection efficiency and other interassay variability. Twenty-four hours later, firefly and Renilla luciferase activities in 10- to 20-µl lysate samples were measured with a Turner Systems 20/20 luminometer (Turner Designs, Sunnyvale, CA) by using the dual-luciferase reporter assay system, following the protocol provided by the manufacturer (Promega, Madison, WI). After background subtraction, firefly luciferase activity was normalized to Renilla luciferase activity in the lysates. The results were recorded as normalized MHK{alpha}2 promoter activity. Each experimental observation represents the mean of results from three independent transfections.

Chromosomal Assignment and Fluorescence In Situ Hybridization
The murine HK{alpha}2 gene was localized by fluorescence in situ hybridization. The {lambda}63a-1 clone was labeled with digoxigenin-11-UTP by nick translation, combined with sheared mouse DNA, and hybridized to normal metaphase chromosomes derived from mouse embryo fibroblasts, in 50% formamide/10% dextran sulfate/2x SSC. Specific hybridization was detected by using hybridization slides with fluorescein-conjugated anti-digoxigenin antibodies, followed by counterstaining with 4,6-diamidino-2-phenylindole. The initial experiment for each probe resulted in specific labeling of the middle region of a medium-size chromosome, which was thought to be chromosome 14 on the basis of 4,6-diamidino-2-phenylindole staining in 59 of 80 metaphase cells. A second experiment was conducted, in which a probe specific for the centromeric region of chromosome 14 cohybridized with the murine HK{alpha}2 gene. Measurements of 10 specifically labeled chromosome 14 copies were performed, for estimation of the distance from the heterochromatic-euchromatic boundary to the telomere of chromosome 14.

Data Analyses
Sequence analyses were performed by using MacVector 6.5.3 software (Genetics Computer Group, Madison, WI). Potential regulatory motifs in the HK{alpha}2 gene were identified with MatInspector version 2.2 (http://www.gsf.de/cgi-bin/matsearch.pl), using the Transfac 5.0 database.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Isolation of Genomic Clones for the Murine HK{alpha}2 Gene
A mouse genomic library constructed using DNA from strain 129Sv/J was screened for clones containing the HK{alpha}2 gene by using a 0.8-kb genomic fragment amplified from mouse tail DNA with primers derived from HK{alpha}2 exons 1 and 2. One clone, {lambda}63a-1, was fully sequenced and found to extend 7.2 kb into the 5'-flanking region and 3' to sequences in intron 8 (Figure 1). The library was rescreened with cDNA probes directed against the 3'-end of rat HK{alpha}2a, to obtain additional genomic clones comprising the 3'-end of the gene. Three additional clones were isolated, i.e., {lambda}4a-1, {lambda}4b-1, and {lambda}5a-2. The complete insert sequence of {lambda}5a-2 was also determined. It spans from exon 6 to exon 23 and has 971 bp overlapping with {lambda}63a-1. Both {lambda}4a-1 and {lambda}4b-1 were partially sequenced; {lambda}4a-1 begins in exon 4 (thus overlapping with {lambda}63a-1 by approximately 2.8 kb) and extends at least to exon 23, and {lambda}4b-1 begins in exon 9 and has a 3.8-kb sequence stretch 3' to exon 23. The locations of exons in the genomic DNA sequence were determined by comparison with the human ATP1AL1 gene and were confirmed with the cDNA sequence. The HK{alpha}2 gene includes 23 exons and 22 introns. A start codon is positioned in the first exon. The GT/AG donor/acceptor consensus site (28) was maintained in all introns. The portions of the clones sequenced (totaling 34 kb) and their corresponding arrangement with respect to the exon/intron structure of the HK{alpha}2 gene are presented in Figure 1. The sequences that flank the exon/intron boundaries in the HK{alpha}2 gene are presented in Table 1. The entire sequence of the murine HK{alpha}2 gene has been submitted to GenBank (accession number AF350499).



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  		Figure 1. Organization and physical mapping of the murine H+/K+-ATPase {alpha}2 (HK{alpha}2) gene. The top line shows the bacterial artificial chromosome (BAC) clone containing the full-length murine HK{alpha}2 gene. {lambda}63a-1, {lambda}5a-2, {lambda}4a-1, and {lambda}4b-1 represent overlapping inserts from {lambda} phage clones and are aligned relative to the BAC clone. Solid boxes indicate the regions that were sequenced. The bottom line shows the numbers and positions of the exons within the gene. Exons are indicated by solid vertical lines and are numbered. The scale is shown at the left. The exact lengths of all exons and introns are presented in Table 1.

 

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  		Table 1. Intron/exon boundaries of the murine HK{alpha}2 genea
 
Isolation of a BAC Clone Containing the Murine HK{alpha}2 Gene
The RPCI-23 BAC library (23) from C57BL6/J mice was used to isolate BAC clones for the HK{alpha}2 gene. This BAC library was constructed in the pBACe3.6 vector (29) at the EcoRI sites. We screened the RPCI-23 BAC library with three separate HK{alpha}2-specific probes generated by overgo PCR from mouse colon cDNA, corresponding to the 5'-UTR, middle, and 3'-end of the coding region of murine HK{alpha}2 cDNA. One clone (RP23-442H19), which hybridized to all three overgo probes at high stringency, was partially sequenced with primers corresponding to the 5'-flanking region and the 3'-UTR of the HK{alpha}2 gene. The sequences in these regions were observed to be identical to the corresponding regions of the HK{alpha}2 gene obtained from the {lambda} clones {lambda}63a-1, {lambda}5a-2, {lambda}4a-1, and {lambda}4b-1 (Figure 1).

Chromosomal Localization of the Murine HK{alpha}2 Gene
The {lambda}63a-1 clone was used for chromosomal localization by fluorescence in situ hybridization analysis, as described in Materials and Methods. A total of 80 metaphase cells were analyzed for each genomic clone, with 59 of 80 chromosomes exhibiting specific labeling for the HK{alpha}2 gene (Figure 2). The HK{alpha}2 gene is positioned 39% of the distance from the heterochromatic-euchromatic boundary to the telomere of chromosome 14, which corresponds to band 14C3.



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  		Figure 2. Chromosomal localization of the murine HK{alpha}2 gene. DNA from {lambda} clones containing genomic regions of the murine HK{alpha}2 gene was labeled with digoxigenin-dUTP and hybridized to normal metaphase chromosomes derived from mouse embryo fibroblasts (for details, see the Materials and Methods section). Cohybridization of the HK{alpha}2 probe (yellow arrow) with a probe specific for the telomeric region on chromosome 14 (white arrow) is shown.

 
Cloning of Murine HK{alpha}2 cDNA
Murine HK{alpha}2 cDNA has not been previously isolated or reported. We used a combination of 5'-RACE, 3'-RACE, and reverse transcription-PCR to clone the entire HK{alpha}2 cDNA from mouse colon cDNA. Anchor-ligated cDNA synthesized from mouse kidney mRNA was subjected to 5'-RACE using adapter primer 2 and a HK{alpha}2-specific reverse primer. A single distinct PCR product of 303 bp was consistently amplified. This product was isolated, subcloned, and sequenced. A total of seven 5'-RACE reactions were analyzed in this manner. In contrast to our studies in rats (17), no alternative 5'-end of the cDNA was identified. Similarly, 3'-RACE yielded a 1.9-kb product that extended to the 3'-end of murine HK{alpha}2 cDNA (not shown). Using primers derived from the 5'- and 3'-ends of this sequence, a 3990-bp cDNA containing the full coding region of HK{alpha}2 was obtained. The deduced sequence of 1036 amino acids of murine HK{alpha}2 was 96, 88, 87, and 86% identical to the sequences of the rat (12,17), guinea pig (14), and rabbit (15) HK{alpha}2 proteins, respectively, and 84% identical to that of the human ATP1AL1 protein. The murine HK{alpha}2 sequence was 77% identical to the sequence for the toad H+/K+-ATPase (30).

Location of the Transcription Start Site and Sequence Analysis of the 5'-Flanking Region
The transcription initiation site for the murine HK{alpha}2 gene was mapped by primer extension analysis of total RNA from the distal colon. A single major extension product was consistently observed (Figure 3), and this corresponded well to the 5'-most ends of the 5'-RACE products from mouse kidney cDNA. Again, the finding of a single primer extension product for murine HK{alpha}2 contrasts with the finding of alternative 5'-splice variants in rats (17) and rabbits (15). The size of the HK{alpha}2 primer extension product places the transcription initiation site 253 bp upstream of the translation initiation methionine codon. The transcription initiation site was labeled as +1 on the genomic DNA (Figure 4). The nucleotide sequences surrounding the HK{alpha}2 transcription initiation site closely match a CAP site consensus sequence (31). The nucleotides surrounding the ATG codon (5'-TCACAGTATGC-3') correspond only weakly to the Kozak consensus sequence for translational initiation (Figure 4).



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  		Figure 3. Primer extension analysis of the 5'-end of murine HK{alpha}2 mRNA. Primer extension experiments were performed with a 32P-labeled, HK{alpha}2-specific, oligonucleotide primer and total RNA from mouse distal colon, as described in the Materials and Methods section. A representative autoradiograph is shown. Yeast tRNA served as a negative control (-). The arrow indicates the position of the major transcription start site. The sizes of the primer extension products were established by comparison with molecular weight standards and with a sequence ladder generated by cycle sequencing with the 32P-labeled primer used for each extension reaction, with a HK{alpha}2 partial genomic DNA clone as template. All experiments were performed in triplicate.

 


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  		Figure 4. Nucleotide sequence and potential cis-elements of the proximal 5'-flanking region of the murine HK{alpha}2 gene. Nucleotides used to generate the oligonucleotide primer for the primer extension assay are indicated by the arrow. A potential TATA-like sequence is indicated by an open box. The transcription initiation site defined in primer extension and 5'-rapid amplification of cDNA ends analyses is indicated in bold type as +1. Sequences exhibiting homology to cis-elements or transcription factor binding sites are underlined. Numbers at the right indicate nucleotide positions, relative to the HK{alpha}2 transcription initiation site.

 
We determined the sequence of 7264 bp of the 5'-flanking region of the murine HK{alpha}2 gene. This region was examined for potential cis-elements that might serve as binding sites for transcription factors. The proximal HK{alpha}2 5'-flanking region contains a TATA-like sequence (ATTTAA) and a CCAAT motif (31), beginning 46 and 152 bp, respectively, 5' to the transcription start site, which likely comprise the core promoter module (Figure 4). Numerous consensus binding sites for transcription factors were observed. These included seven Sp1 sites (32), 36 activator protein-1 sites, 49 GATA-1 sites (33), 16 CCAAT enhancer-binding protein sites (34), three nuclear factor-{kappa}B sites (35), four cAMP-responsive element-binding protein sites, and four gut-enriched Kruppel-like factor (GKLF) motifs (36), as determined using a core similarity score of >0.90 and a matrix similarity score of >0.90 in the MatInspector version 2.2 program. In addition, two potential glucocorticoid response elements (GRE) were identified, i.e., 5'-GGATCAAACTGTCC-3', beginning at -6598 (core similarity score, 0.923; matrix similarity score, 0.897), and 5'-CAGAACAGCTGGTAAA-3', beginning at -1071 (core similarity score, 1.00; matrix similarity score, 0.907).

Tissue Distribution of Murine HK{alpha}2 mRNA
Northern blots of total RNA collected from an array of mouse tissues were probed with a 32P-labeled DNA probe specific for HK{alpha}2 (Figure 5). A single major band of approximately 4 kb, which corresponds well to the full-length murine HK{alpha}2 cDNA determined in 5'- and 3'-RACE and primer extension analyses, was detected. HK{alpha}2 was expressed prominently in the distal colon and weakly in normal kidney, brain, and uterus. The expression in kidney, brain, and uterus was more evident with longer radiographic exposures. No appreciable HK{alpha}2 mRNA expression was detected in spleen, liver, or ileum. Staining of the gels or blots for 28S and 18S RNA indicated equal loading and transfer among samples (data not shown).



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  		Figure 5. Distribution of HK{alpha}2 mRNA in mouse tissues. A Northern blot of total RNA was probed with a 32P-labeled cDNA probe specific for murine HK{alpha}2. Staining of 18S and 28S RNA indicated equal integrity, loading, and transfer among the samples (data not shown). U, uterus; C, distal colon; K, kidney; I, ileum; B, brain; L, liver; S, spleen.

 
Collecting Duct-Selective Activity of the HK{alpha}2 Promoter
Despite the fact that this gene transcriptionally responds to important physiologic stimuli to effect adaptive changes in K+ transport, to date there have been no published studies examining the function of any HK{alpha}2 promoter, including ATP1AL1. To determine whether the proximal 5'-flanking region of the HK{alpha}2 gene contained a functional gene promoter, transient expression in cultured cells was performed. A fragment of the genomic clone {lambda}63a-1 containing the transcription initiation site and 7.2 kb of proximal 5'-flanking region was cloned upstream to a promoterless luciferase reporter gene in the plasmid pGL3-Basic. The resulting plasmid (designated pGL3-7.2MHK{alpha}2) was transfected into mIMCD3 or mOMCD1 collecting duct cells, ST-1 MTAL cells, or NIH 3T3 fibroblasts. As a control for transfection efficiency, cells were cotransfected with pRL-SV40 and Renilla luciferase activity was measured. Promoter activity was inferred from light output, normalized for differences in transfection efficiency (which were minimal). Transfection with pGL3-Basic produced low levels of luciferase in all four cell types. Figure 6 presents the light output after transfection of mIMCD3, mOMCD1, ST-1, and NIH 3T3 cells with pGL3-7.2MHK{alpha}2, normalized for each cell line to that of pGL3-Basic. Transfection with pGL3-7.2MHK{alpha}2 produced minimal luciferase in NIH 3T3 fibroblasts and ST-1 cells but considerably more luciferase (approximately eight- to tenfold higher levels) in mOMCD3 and mIMCD3 cells. Promoter activity was greatest in mOMCD3 cells, in keeping with the fact that the OMCD is the principal intrarenal site of HK{alpha}2 expression in vivo. Taken together, these results demonstrated that the cloned 7.2-kb fragment contained a promoter that was highly active in renal collecting duct cells and that, among the cell types tested, the HK{alpha}2 promoter exhibited collecting duct-selective activity.



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  		Figure 6. Activity of the murine HK{alpha}2 gene promoter in cultured cell lines. The renal collecting duct cell lines mIMCD3 and mOMCD1, which are known to express HK{alpha}2, and NIH 3T3 cells and ST-1 medullary thick ascending limb of Henle’s loop (MTAL) cells, which do not express HK{alpha}2, were transiently cotransfected with pGL3-7.2MHK{alpha}2 (which contains the proximal 7.2 kb of the murine HK{alpha}2 promoter fused to the luciferase gene) or the promoterless parent vector pGL3-Basic and pRL-SV40 (to normalize for transfection efficiency). Firefly and Renilla luciferase activities in lysates of the cells were then determined with a luminometer, and the ratios were recorded. The relative activity of the HK{alpha}2 promoter constructs, compared with pGL3-Basic luciferase activity, in each cell line is plotted. The means (bars) of three to five separate experiments are shown.

 
Deletion Analysis of the 5'-Flanking Region of the HK{alpha}2 Gene
To identify the locations of regulatory elements required for HK{alpha}2 promoter activity in collecting duct cells, promoter deletion analysis was performed. Luciferase reporter plasmids that contained a set of deletions extending from nucleotide +253 within the first exon to various positions within the 5'-flanking region were constructed. Plasmids were transfected into mIMCD3, mOMCD1, and ST-1 MTAL cells, and normalized luciferase activities were measured. As demonstrated in Figure 7, luciferase activities were high and quite similar for the collecting duct cell lines transfected with the promoter with deletions from -7265 to -177. In contrast, the deletion constructs exhibited low-level activity when transfected into ST-1 MTAL cells. These results indicate that positive regulatory elements that control HK{alpha}2 promoter activity in renal collecting duct cells are likely to be located within the proximal 0.177 kb of the 5'-flanking region. The data also indicate that the minimal promoter for the murine HK{alpha}2 gene must be contained within the proximal 0.177 kb of the 5'-flanking region.



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  		Figure 7. Deletion analysis of the murine HK{alpha}2 gene promoter in cultured renal cell lines. mIMCD3, mOMCD1, and ST-1 MTAL cells were transfected with pGL3.177MHK{alpha}2, pGL3.477MHK{alpha}2, pGL3-1.3MHK{alpha}2, pGL3-2.8MHK{alpha}2, pGL3-4.3MHK{alpha}2, pGL3-5.9MHK{alpha}2, or pGL3-7.2MHK{alpha}2 (containing various lengths of the murine HK{alpha}2 promoter) or the promoterless parent vector pGL3-Basic, together with pRL-SV40 (to normalize for transfection efficiency). The mIMCD3 and ST-1 cells were transfected with all constructs, whereas the mOMCD1 cells were transfected with all except the pGL3.177MHK{alpha}2 and pGL3.477MHK{alpha}2 constructs. Firefly and Renilla luciferase (LUC) activities in lysates of the cells were then determined with a luminometer, and the ratios were recorded. The relative activity of the HK{alpha}2 promoter constructs, compared with pGL3-Basic luciferase activity, in each cell line is plotted. The means (bars) of three to five separate experiments are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
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 regarding potential control mechanisms governing HK{alpha}2 gene expression. In this study, we isolated and sequenced the murine HK{alpha}2 gene, including all introns and exons and an extended stretch of the 5'-flanking region. In addition, we localized the transcription initiation site and assigned the gene to mouse chromosome 14C3. This structural information should prove to be of fundamental importance in the design and interpretation of studies examining the regulation of the HK{alpha}2 gene in response to provocative stresses, such as chronic hypokalemia or changes in the aldosterone balance. The isolation of a BAC clone harboring the murine HK{alpha}2 gene, together with the {lambda}63a-1 clone containing the 5'-flanking region of the gene, provides necessary reagents for investigation of the transcriptional regulation of the HK{alpha}2 gene in transgenic mice. In addition, we provide the first evidence of functional promoter activity for any HK{alpha}2 gene family member, by demonstrating that the proximal 5'-flanking region of the murine HK{alpha}2 gene is functionally active in murine collecting duct cells.

The HK{alpha}2 gene seems to be differentially expressed in rodent kidney and distal colon under basal conditions and in response to hypokalemia and excess aldosterone. Under basal conditions, the gene is robustly expressed in rat distal colon but is more weakly expressed in the renal medulla. After K+ deprivation, however, the HK{alpha}2 gene is upregulated in rat and mouse kidney outer medulla but not in distal colon (17). Our group (13) and others (37) have demonstrated that the HK{alpha}2 gene is greatly upregulated, principally in the OMCD and to a lesser extent in the proximal IMCD, in rats. In contrast to the upregulation of the HK{alpha}2 gene observed in medullary collecting ducts, four (8,13,17,37) of five studies (8,13,17,37,38), including our own, indicated that HK{alpha}2 expression is unaltered in rat distal colon during chronic K+ deprivation. Further studies to examine the responsiveness to low external K+ concentrations of the HK{alpha}2 promoter expressed in the collecting duct cell lines and in relevant colonic epithelial cell lines should help clarify the transcriptional basis for these differential responses.

Aldosterone and chronic Na+ depletion (which induces a state of secondary hyperaldosteronism) promote two- to threefold increases in active K+ absorption by the rat distal colon mucosa (38,39). More recent studies demonstrated that chronic Na+ depletion in rats stimulated a 3.5-fold increase in HK{alpha}2 protein expression and a twofold increase in ouabain-insensitive H+/K+-ATPase activity in apical membranes from the colon (7). Consistent with these findings, adrenalectomy resulted in reduced HK{alpha}2 gene expression in the rat distal colon, and expression was restored to normal levels by exogenous administration of aldosterone (8). Chronic Na+ depletion results in other adaptive responses, but existing data suggest that the principal effects on HK{alpha}2 expression and activity in this tissue during chronic Na+ deprivation are mediated by excess aldosterone; colonic Na+ and K+ transport is identical in rats receiving continuous, high-level aldosterone infusions and rats subjected to chronic Na+ deprivation (39), and serum aldosterone levels are comparable in dietary Na+-depleted animals and those infused with aldosterone via minipumps (40). In contrast to the colon, aldosterone does not alter HK{alpha}2 expression in the kidney (8).

Our finding of two potential GRE in the 5'-flanking region of the murine HK{alpha}2 gene, to which liganded mineralocorticoid receptors might bind, provides a possible molecular explanation for the known effects of aldosterone in stimulating the HK{alpha}2 gene in vivo. Of the X+/K+-ATPase {alpha} subunits, a functional composite GRE has recently been identified in the 5'-flanking region of the Na+/K+-ATPase {alpha}1 subunit (41). The sequence and position of the GRE in the murine HK{alpha}2 gene are dissimilar from those of the GRE in the Na+/K+-ATPase {alpha}1 gene. Many other consensus sites for transcription factor binding were also identified in the murine HK{alpha}2 gene 5'-flanking region. Interestingly, a number of binding motifs for GKLF were identified. GKLF is a zinc finger-containing transcription factor (42) that is enriched in gastrointestinal epithelial cells, where it resides in the middle to upper crypts of the colonic mucosa. It represents a candidate factor that might regulate HK{alpha}2 expression in the distal colon, which might explain the high basal rates of HK{alpha}2 expression in that organ. No cis-elements involved in the transcriptional response to chronic hypokalemia have been reported for any X+/K+-ATPase gene. The fact that a deletion construct containing only the proximal 0.177 kb of the murine HK{alpha}2 promoter was functional in renal collecting cell lines known to express HK{alpha}2 but was much less responsive in the MTAL cell line, which does not express HK{alpha}2, suggests that sequence information within this proximal promoter region confers the transcriptional response in renal collecting duct cells.

The chromosomal location of murine HK{alpha}2 is distinct from the locations of other {alpha}-subunits of the Na+/K+-ATPase/H+/K+-ATPase gene family. The gastric H+/K+-ATPase {alpha}1 and Na+/K+-ATPase {alpha}3 subunits are closely linked on mouse chromosome 7 (43). The Na+/K+-ATPase {alpha}1 subunit gene resides on mouse chromosome 3, whereas the Na+/K+-ATPase {alpha}2 and {alpha}4 subunit genes reside on mouse chromosome 1 (44).

Whether the HK{alpha}2 forms cloned from nonhuman mammals represent orthologs or unique isoforms of the human ATP1AL1 subunit has been controversial. Our structural and sequence data strongly suggest that the murine HK{alpha}2 and human ATP1AL1 genes are orthologous. The murine HK{alpha}2 gene maps to mouse chromosome 14C3, which seems to be syntenic with the location of human ATP1AL1. The two genes share a common exon/intron structure and significant sequence homology in the coding region and 3'-UTR. This commonality of structural organization extends to other members of the X+/K+-ATPase multigene family. The HK{alpha}2, ATP1AL1, and Na+/K+-ATPase {alpha} subunit genes all consist of 23 exons. However, comparison of the 5'-flanking region of the murine HK{alpha}2 gene with those of ATP1AL1 and genes for the Na+/K+-ATPase {alpha}1 subunit (45), Na+/K+-ATPase {alpha}2 subunit (46), and Na+/K+-ATPase {alpha}3 subunit (47) demonstrated no significant homologies. The CpG islands observed in the ATP1AL1 5'-flanking region are absent from the murine HK{alpha}2 gene.

The data presented in this report should prove useful in functional studies of potential cis-elements governing differential transcriptional regulation of the HK{alpha}2 gene. Because such gene control elements might be dispersed throughout the gene, knowledge of the complete gene sequence, as reported here, should facilitate the discovery of HK{alpha}2 gene control mechanisms.


   Acknowledgments
 
This work was supported by National Institutes of Health Grant R01-DK47981 and was conducted during the tenure of Dr. Kone as an Established Investigator of the American Heart Association. We acknowledge the expert assistance of Dr. David Nelson and Sali Shaydel, Baylor College of Medicine (Houston, TX), and the gift of the RPCI-23 library from Dr. Richard Gibbs, Baylor College of Medicine. Sequence data from this article have been deposited in the GenBank/EMBL data libraries (accession number AF350499).


   References
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 Introduction
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 Results
 Discussion
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Received for publication May 3, 2001. Accepted for publication June 18, 2001.




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