A Novel N-terminal Splice Variant of the Rat H+-K+-ATPase α2 Subunit

The H+-K+-ATPase of renal collecting duct mediates K+ conservation during chronic hypokalemia. K+ deprivation promotes H+-K+-ATPase α2 (HKα2) 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 HKα2 subunit. Sequence analysis andin vitro translation revealed that HKα2a corresponds to the known HKα2 cDNA (Crowson, M. S., and Shull, G. E. (1992) J. Biol. Chem. 267, 13740–13748), whereas HKα2b represents a novel variant truncated by 108 amino acids at its N terminus. HKα2b mRNA contains a complex 5′-untranslated region with eight upstream open reading frames, features implicated in translational regulation of other genes. Heterologous expression of HKα2b with and without the gastric H+-K+-ATPase β 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 β subunit for optimal functional activity. Northern analysis demonstrated that both subtypes (HKα2b > HKα2a) 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 HKα2 gene expression by transcriptional and posttranscriptional mechanisms not recognized in other members of the Na+-K+-ATPase/H+-K+-ATPase family.

The maintenance of body potassium (K ϩ ) balance is critical to the normal function of all cells. Perturbations in K ϩ 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 ϩ balance in mammals, adjusting K ϩ excretion to match large variations in dietary K ϩ intake. The late distal tubule and collecting duct have the dual ability to secrete and reabsorb K ϩ as needed to effect this balance (1,2). In response to chronic dietary K ϩ deprivation, these segments, in particular the OMCD 1 actively reclaim filtered K ϩ . Physiological, biochemical, and molecular biological studies have shown that this adaptation is principally attributable to increased expression and/or activity of an H ϩ -K ϩ -ATPase in the luminal membrane of these segments (2)(3)(4)(5)(6)(7)(8)(9). A similar transport system(s) has been identified in the apical membrane of mammalian distal colon, where it, too, effects active K ϩ absorption (10,11). Although active K ϩ absorption in the distal colon is enhanced during K ϩ depletion (11) and participates to a limited degree in restoring K ϩ balance, the identity of the specific K ϩ -ATPase that is up-regulated remains controversial (12).
The H ϩ -K ϩ -ATPases constitute a subfamily of isozymes that belong to the X ϩ -K ϩ -ATPase multigene family, which also includes the Na ϩ -K ϩ -ATPase isoforms. The X ϩ -K ϩ -ATPases share common catalytic and ion transport mechanisms and an apparent requirement for heterodimeric (␣:␤) structure. The X ϩ -K ϩ -ATPase ␣ subunits exhibit considerable (ϳ65%) 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 ϩ -K ϩ -ATPase ␣ subunits have been cloned from mammals, structurally characterized, and expressed in heterologous systems. The H ϩ -K ϩ -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 ϩ -K ϩ -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)(6)(7)(8). Expression of the HK␣2 subunit with the known rat X ϩ -K ϩ -ATPase ␤ subunits (16) or toad bladder H ϩ -K ϩ -* The costs of publication of this article were defrayed in part by the payment of page charges. This 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 GenBank TM  ATPase ␤ subunit (18) in Xenopus laevis oocytes resulted in the appearance of active H ϩ -K ϩ 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 ϩ -ATPase activity was Sch 28080-and ouabain-resistant (19). A third H ϩ -K ϩ -ATPase ␣ subunit cDNA, termed ATP1AL1 (or H ϩ -K ϩ -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 ϩ -K ϩ -ATPase ␤ subunit (HK␤ g ) in Xenopus oocytes (21) or HEK 293 cells (22) resulted in the expression of functional H ϩ -K ϩ pumps that were partially sensitive to both Sch 28080 and ouabain.
Recent studies by our laboratory and others have shown that chronic K ϩ deprivation enhances HK␣2, but not HK␣1 (3), gene expression in the OMCD (5)(6)(7)(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 ϩ -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 ϩ 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 HK␣2 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 ϩ -K ϩ -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 ϩ deprivation.
Oligonucleotide Primers-PCR primers not included in specific kits were synthesized by Genosys, Inc. (The Woodlands, TX). The sequences of the various HK␣2 subunit primers are presented in Fig. 1A, and those of the HK␤ g subunit are given below.
5Ј-RACE and Cloning of H ϩ -K ϩ -ATPase ␣2b cDNA-The 5Ј-RACE protocol was performed using the Marathon TM cDNA Amplification Kit (CLONTECH, Palo Alto, CA), according to the manufacturer's instructions. First strand cDNAs were generated from 1 g of rat kidney poly(A) ϩ RNA, using Moloney murine leukemia virus reverse transcriptase and a modified locking oligo(dT) primer containing two degenerate nucleotide positions at its 3Ј 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 5Ј-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 ϩ344 to ϩ325 of the published HK␣2 cDNA sequence (17), and the components of the Advantage TM cDNA Amplification Kit (CLONTECH). PCR cycling conditions were as follows: 94°C ϫ 1 min, followed by 28 cycles of 94°C ϫ 30 s, 68°C ϫ 4 min, and a final step of 68°C ϫ 4 min. Ten l 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 pCR2.1 TM (Invitrogen) and sequenced on both strands by a cycle sequencing method.
To establish the coding sequences 3Ј to the alternative splice site of the HK␣2b variant (see "Results"), the complete encoding DNA was PCR-amplified from oligo(dT) 17 -primed rat kidney cDNA, using the HK␣2b-specific sense primer P5 (Fig. 1A) and a common antisense primer (P10, 5Ј-GCTCGAGGAATCATAGTCTAGC-3Ј) located in the 3Ј-UTR (nucleotides 3647-3667) of the published HK␣2 sequence (17). An XhoI site (underlined) was incorporated into the 5Ј end of the primer to facilitate eventual subcloning into the mammalian expression vector pcDNA3.1Ϫ/Neo (Invitrogen). The amplicons were first subcloned into pCR2.1 TM and sequenced on both strands. The sequence-verified encoding DNA for HK␣2b was then excised from pCR2.1 TM and cloned into the XbaI and XhoI sites of pcDNA3.1Ϫ/Neo downstream of the cytomegalovirus promoter. The resultant recombinant molecule was designated pcDNA3.1Ϫ/HK␣2b-Neo.
Cloning of the Rat HK␤ g 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 5Ј-ATAAGCTTCAGCCCTG-CAGGAGAAG-3Ј (nucleotides ϩ16 to ϩ32 of the published sequence (25)) and antisense 5Ј-ATTCTAGATTACTTCTGTATTGTGAGC-3Ј (nucleotides ϩ878 to ϩ896 of the published sequence). HindIII and XbaI sites (underlined) were added to the 5Ј 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 pcDNA3.1ϩ/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 HK␣2a (P7) and HK␣2b (P8 and P9) were 5Ј-end-labeled with [␥-32 P]ATP using T4 polynucleotide kinase. The primers were annealed to 10 g 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 MgCl 2 , 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 32 P-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 5Ј end of the HK␣2 gene was analyzed using the Rat PromoterFinder TM DNA Walking Kit (CLON-TECH), 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 ϫ 25 s, 72°C ϫ 4 min for 7 cycles, 94°C ϫ 25 s, 67°C ϫ 4 min for 32 cycles, and 67°C ϫ 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 H 2 0 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 ϫ 25 s, 72°C ϫ 4 min for 7 cycles, 94°C ϫ 25 s, 67°C ϫ 4 min for 20 cycles, and 67°C ϫ 4 min for 1 cycle. The amplified products were separated by electrophoresis in a 0.9% agarose gel, subcloned into pCR2.1 TM , 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 ϩdeprived 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 HK␣2a and HK␣2b cDNAs, using primer pairs P3 ϩ P4 and P5 ϩ P6 (Fig. 1A) directed at the unique 5Ј 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 32 P by the random primer method according to the manufacturer's instructions (Prime-a-Gene, Promega, Madison, WI). Fifteen g 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 ϫ SSC, 0.1% (s/v) SDS at 60°C. Autoradiographs of the blots were prepared at Ϫ70°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-pcDNA3.1Ϫ/HK␣2b-Neo and the HK␣2a encoding DNA subcloned into the vector pAGA2 (16) were transcribed and translated in the presence of [ 35 S]methionine with T7 RNA polymerase and the TNT-coupled reticulocyte lysate kit (Promega, Madison, WI). The synthesized proteins were separated by SDSpolyacrylamide 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 g/ml streptomycin, 5 g/ml insulin, 5 g/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 pcDNA3.1Ϫ/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 g of plasmid DNA and 22 l 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% CO 2 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 g/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 g/ml G418 and 250 g/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.
86 Rb ϩ Uptake-Uptake of 86 Rb ϩ , a K ϩ 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 MgCl 2 , 1 mM CaCl 2 , 2 mM NaH 2 PO 4 , 32 mM HEPES, pH 7.4, and 200 M 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 ϩ was used in these assays, because K ϩ competitively inhibits both Sch 28080 and ouabain binding to X ϩ -K ϩ -ATPase ␣ subunits, and this concentration is within the narrow range of K m values reported for K ϩ dependence of all known X ϩ -K ϩ -ATPase ␣ subunits. Uptake was initiated by adding 0.2 ml of uptake buffer containing ϳ4 Ci/ml 86 Rb ϩ . After 12 min, the reaction was stopped by six rapid washes with ice-cold stop buffer (100 mM MgCl 2 , 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 86 Rb ϩ 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 HK␣2 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 Ϯ S.E. and were analyzed for significance by analysis of variance. Significance was assigned at p Ͻ 0.05.

RESULTS
cDNA Cloning and Structural Analysis of a Truncated Nterminal Variant of the H ϩ -K ϩ -ATPase ␣2 Subunit-The anchor-ligated cDNAs synthesized from rat kidney mRNA were subjected to 5Ј-RACE using adapter primer 2 and HK␣2-specific primer P1 from exon 2 (Fig. 1A). Two distinct PCR products of ϳ400 and ϳ600 bp, subsequently shown to correspond to the 5Ј 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 5Ј ends but were identical at their 3Ј ends, with common sequence beginning at the codon for Lys 4 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 5Ј end, so that the total 5Ј-UTR was 274 bp.
By using a sense primer from the 5Ј-UTR of HK␣2b and an antisense primer derived from the 3Ј-UTR of the published HK␣2 cDNA (17), a 3847-bp cDNA, including the entire HK␣2b coding region, was PCR-amplified from rat kidney cDNA, subcloned, and sequenced. The HK␣2b sequence was identical to that of HK␣2a beginning at the codon for Lys 4 of HK␣2a (Fig.  1A). The first AUG triplet of the HK␣2b mRNA that resides within a favorable context for translation initiation corresponds to Met 109 of HK␣2a. Thus the predicted HK␣2b peptide of 929 amino acids (mass ϭ 102,554 Da) lacks the first 108 amino acids of the HK␣2a sequence (1036 amino acids, mass ϭ 114,966 Da), which includes consensus sites for cAMP phosphorylation (Thr 5 ) and protein kinase C phosphorylation (Ser 78 ) (Figs. 1A and 2). Secondary structure models of the HK␣2a and HK␣2b deduced amino acid sequences predict that HK␣2b would have a shorter N-terminal cytosolic segment but would otherwise share identical topology to HK␣2a (Fig. 2).
As predicted from the sequence analysis, in vitro transcription and translation of HK␣2b cDNA yielded an ϳ104-kDa protein, whereas in vitro transcription and translation of HK␣2a cDNA yielded ϳ118and ϳ104-kDa proteins (Fig. 3). The latter result indicates that both HK␣2 variant proteins can be translated in vitro from HK␣2a mRNA by utilization of the first and second in-frame AUG codons.
Genomic Organization of the 5Ј End of the HK␣2 Gene and Mapping of the Transcription Start Sites-Analysis of the HK␣2a and HK␣2b 5Ј-RACE products suggested that both mRNAs are derived from a single gene by utilization of alternative splice sites at the 5Ј region. To determine the order of the exons and the intervening genomic sequences, we used nested reverse primers derived from the 5Ј region common to both variants and adapter-ligated rat genomic DNA libraries to PCR amplify a portion of the 5Ј region of the HK␣2 gene. PCR products of ϳ1.6and 0.5-kb were consistently amplified from the DraI and PvuII libraries, respectively. These amplicons were subcloned into pCR2.1 TM 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 5Ј end of the HK␣2b mRNA represents a 5Ј extension of exon 2 that is excised in the HK␣2a mRNA. In support of this construct, a consensus 5Ј splice donor site (5Ј-GTGAGT-3Ј) was identified at the exon 1/intron 1 boundary, and a consensus 3Ј acceptor site (CAG) (29) was found in the expected 5Ј region of exon 2 (Fig.  1A).
The transcription initiation sites for the two mRNAs were mapped by primer extension analysis of total RNA from distal colon. A single major extension product was observed for both the HK␣2a and HK␣2b reactions (Fig. 4), and these corresponded within 2 to 3 nucleotides to the 5Ј-most ends of the 5Ј-RACE products from rat kidney cDNA. The size of the HK␣2a primer extension product places the transcription initiation site 274 bp upstream of the initiation methionine codon, the first ATG triplet 3Ј to the transcription start site (Fig. 1A). The nucleotide sequences surrounding the HK␣2a transcription initiation site closely matches a CAP site consensus sequence (30). The putative transcription initiation site for HK␣2b resides 424 bp upstream of the exon 1/exon 2 alternative splice junction. The total length of the HK␣2b 5Ј-UTR is 739 bp, and the length of the mRNA characterized is 3874 bp. Interestingly this 5Ј-UTR region contains eight upstream open reading frames (uORFs) as follows: 1) ϩ24 to 68; 2) ϩ117 to ϩ446; 3) ϩ139 to ϩ315; 4) ϩ189 to ϩ446; 5) ϩ489 to ϩ560; 6) ϩ579 to ϩ773; 7) ϩ618 to ϩ773; and 8) ϩ678 to ϩ773 (numbering with ϩ1 at putative HK␣2b transcription start site).
Analysis of Potential Gene Control Elements-The HK␣2 partial genomic clone contained sequences of ϳ380 and ϳ205 bp immediately 5Ј 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 5Ј-flanking region contains a TATA-like sequence (ATTTAA), a CACCC sequence (31), and a CCAAT motif (30) beginning 27, 127, and 133 bp, respectively, 5Ј 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).
The region 5Ј to the transcription start site of HK␣2b contains potential promoter elements, including a TATATAT motif, a reverse complement of a CCAAT sequence, and a CACCC sequence 74, 61, and 120 bp, respectively, upstream of the putative transcription start site. Two Sp1 sites, two AP-2 sites, and single sites for NF-interleukin 6 (39), IRF-1 (40), and GATA-1 were identified in the 5Ј-flanking region of the HK␣2b transcription unit (Fig. 1A).
HK␣2 Isoform mRNAs Are Expressed in Colon and Kidney-Northern blots of total RNA harvested from an array of tissues harvested from K ϩ -replete rats were probed with 32 P-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 mus- cle, 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 ϩ -K ϩ -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 32 P-labeled DNA probe for GAPDH indicated comparable abundance and integrity of the blotted RNA samples (data not shown).
Expression of HK␣2 Subunit Isoform mRNAs in Kidney and Colon of Control and K ϩ -restricted Rats-To determine the response of the HK␣2a and HK␣2b gene products to dietary K ϩ restriction in rat kidney and colon, Northern analysis with 32 P-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 ϩ -restricted rats. In control rats, an abundant ϳ4.0-kb subunit transcript (HK␣2b Ͼ Ͼ 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 ϳ6.0-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 3Ј-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 ϳ4.0-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.
To determine whether the relative levels of the HK␣2a or HK␣2b subunit mRNAs in kidney and colon varied with body K ϩ balance, Northern blots of total RNA isolated from control and K ϩ -restricted rats (n ϭ 4 for each group) were probed sequentially with the subtype-specific probes of comparable size and specific activity. Autoradiographs of the blots prepared after 3 days of film exposure (to allow detection of HK␣2a mRNA in controls) were analyzed by scanning densitometry. The K ϩ -restricted rats exhibited greater levels of both HK␣2a and HK␣2b in the cortex and outer and inner medulla compared with controls (Fig. 6A). The two subtypes appeared to be coordinately up-regulated in the kidney zones of K ϩ -restricted rats, but accurate quantitation of the degree to which expression was enhanced with chronic K ϩ deprivation was not possible because of the low basal expression of both mRNAs in the kidney. For each K ϩ -restricted animal, the abundance of HK␣2b mRNA was greater than that of HK␣2a in each renal parenchymal zone (Fig. 6B), although the magnitude of the difference was highly variable. In contrast to kidney, neither the HK␣2a nor HK␣2b transcript abundance in proximal or distal colon differed between control and K ϩ -restricted rats FIG. 4. Primer extension analysis of 5 ends of H ؉ -K ؉ -ATPase ␣2 isoform mRNAs. Primer extension experiments were performed with 32 P-labeled HK␣2 isoform-specific oligonucleotide primers P7 (HK␣2a) and P8 and P9 (HK␣2b, see Fig. 1A), respectively, and total RNA from rat distal colon as described under "Experimental Procedures." Representative autoradiographs for the HK␣2a and HK␣2b (primer P9 results) results are shown in A and B, respectively. Identical mapping results were obtained with the HK␣2b primers P8 and P9. Yeast tRNA served as a negative control (Ϫ). Lanes 1-5 represent RNA samples obtained from 5 different rats. Lanes A, C, G, and T are sequencing reactions on the same gel using the same primer and the plasmid construct bearing the HK␣2 1.6-kb genomic fragment obtained from the DraI-digested rat genomic DNA library (see "Experimental Procedures"). The base corresponding to the major transcription start site for each isoform is labeled by an asterisk within the genomic DNA sequence shown to the right. The slightly slower mobility of the primer extension products in lanes 3-5 of A reflects a slight delay in loading of these samples. In both figures, short and long exposures of the film were used to allow optimal comparison of the sequencing ladders with the primer extension reactions (which were run on the same gel). All experiments were performed in triplicate.
Functional Expression of the H ϩ -K ϩ -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 ϩ -K ϩ -ATPase ␣ or ␤ subunit gene products, their endogenous Na ϩ -K ϩ -ATPase is highly sensitive to ouabain (22), and they permit analysis of H ϩ -K ϩ -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).
Northern analysis revealed that cells stably transfected with the DNA encoding HK␣2b (HEK-HK␣2b cells) expressed the expected ϳ4.0-kb mRNA recognized by the HK␣2b probe (Fig.  7A). The HEK-NEO and HEK-HK␣2b cells were then stably transfected with the HK␤ g cDNA. Northern analysis revealed that the resulting HEK-HK␤ g cells (data not shown) and the HEK-HK␣2b/HK␤ g cells expressed the ϳ1.4-kb transcript expected for the HK␤ g mRNA containing the bovine growth hormone poly(A) tail provided by the pcDNA3.1ϩ/Zeo vector (Fig.  7B). In contrast, HEK-NEO cells exhibited neither HK␣2b (Fig.  7A) nor HK␤ g (Fig. 7B) gene expression.
As an initial characterization of the functional properties of the HK␣2b subunit, untransfected HEK 293, HEK-NEO, HEK-HK␣2b, and HEK-HK␣2b/HK␤ g cells were grown in media containing 1 M ouabain. Only the HEK-HK␣2b/HK␤ g cell lines survived ouabain treatment, suggesting that the fully assembled HK␣2b/HK␤ g pump can compensate for an inoperative Na ϩ -K ϩ -ATPase in maintaining the intracellular ionic milieu, as has been reported for the ATP1AL1/HK␤ g pump (22). 86 Rb ϩ uptake of HEK-NEO, HEK-HK␤ g , HEK-HK␣2b clone 25, and HEK-HK␣2b/HK␤ g clone 40 cell lines was assayed to determine whether the truncated variant could be expressed in the plasma membrane to conduct active K ϩ uptake. Bumetanide was included in the incubation medium to inhibit K ϩ entry via the Na ϩ -K ϩ -2Cl Ϫ transporter. The basal rate of uptake, measured in the absence of ouabain, was comparable among the different cell lines, with the exception of the HEK-HK␣2b clone 25 cells, whose basal uptake was ϳ20% less (p Ͻ 0.05) than the other transfectants: (in nmol/mg protein/min; n ϭ 3 for each) HEK-NEO, 4.8 Ϯ 0.05; HEK-HK␤ g , 4.2 Ϯ 0.06; HEK-HK␣2b, 3.3 Ϯ .01; HEK-HK␣2b/HK␤ g , 4.2 Ϯ 0.7. As seen in Fig. 8A, the endogenous Na ϩ -K ϩ -ATPase of the wild-type HEK 293 and HEK-NEO cells was quite sensitive to ouabain inhibition as follows: 1 M inhibited ϳ97% of the total 86 Rb ϩ uptake, and 1 mM ouabain virtually abolished uptake in the presence of external 1 mM K ϩ . Similar sensitivity to ouabain inhibition was observed in HEK-HK␤ g cells (Fig. 8A). In contrast, the HEK-HK␣2b clone 25 and HEK-HK␣2b/HK␤ g clone 40 cell lines were less sensitive to 1 M ouabain, exhibiting uptakes that were ϳ3.5and 5-fold greater, respectively, than the HEK-NEO control (Fig. 8A). In the presence of 1 mM oua-

expression in colon and renal parenchymal zones of control (C) and potassium-restricted (2) rats.
A, representative Northern blot of total RNA from cortex (CTX), outer medulla (OM), inner medulla (IM), proximal colon (PC), and distal colon (DC) isolated from control and K ϩ -restricted rats (n ϭ 4 animals for each group). The filters were probed sequentially with 32 P-labeled DNA probes specific for HK␣2a, HK␣2b, and GAPDH as described under "Experimental Procedures." Autoradiographic exposure was overnight. B, histogram showing results of densitometric analysis of Northern blots. The ratio of the relative optical density of the HK␣2b and HK␣2a transcript bands in the K ϩ -restricted rats is plotted. FIG. 7. Heterologous expression of the H ؉ -K ؉ -ATPase ␣2b subunit with or without the gastric H ؉ -K ؉ -ATPase ␤ subunit (HK␤ g ) in HEK 293 cells. A, autoradiograph of representative Northern blots of total RNA harvested from HEK-NEO cells (NEO) and HEK-HK␣2b (2b) clone 25 cells. The blot was probed with a 32 P-labeled DNA probe specific for HK␣2b. B, autoradiograph of Northern blot of total RNA harvested from HEK-NEO and HEK-HK␣2b/HK␤ g (␤ g ) clone 40 cells. The latter cell line was generated by stable transfection of HEK-HK␣2b clone 25 cells with the DNA encoding HK␤ g . The blot was probed with a 32 P-labeled DNA probe specific for HK␤ g . The minor, higher molecular weight bands presumably represent processing intermediates or differences in polyadenylation (provided by the bovine growth hormone poly(A) sequence included in the pcDNA3.1ϩ/Zeo vector) of the HK␤ g mRNA.
bain, 86 Rb ϩ uptake by the HEK-HK␣2b clone 25 and HEK-HK␣2b/HK␤ g clone 40 cell lines was roughly 1.5-to 2-fold greater than the HEK-NEO controls (Fig. 8A). Dose-response curves for ouabain inhibition of 86 Rb ϩ uptake (Fig. 8B) con-firmed that the HK␣2b/HK␤ g clone 40 cells contributed two components of 86 Rb ϩ uptake: one that was extremely sensitive to ouabain (the endogenous Na ϩ -K ϩ -ATPase) and one that was intermediate in its sensitivity to ouabain (the HK␣2b pump). Assuming, then, that the 86 Rb ϩ uptake mechanism that operates in the presence of Ն1 M ouabain in these cells represents the contribution of the HK␣2B pump, the approximate IC 50 (IC 50 , concentration of inhibitor causing 50% inhibition of corresponding 86 Rb ϩ uptake) for ouabain inhibition of 86 Rb ϩ uptake for the HK␣2B pump was ϳ400 to 800 M in the presence of external 1 mM K ϩ (Fig. 8B).
The effects of Sch 28080, a potent inhibitor of the HK␣1 subunit (16), were tested on the component of 86 Rb ϩ uptake insensitive to 1 M ouabain. Sch 28080, at concentrations up to 500 M, had no statistically significant effect on 86 Rb ϩ uptake in the HEK-NEO, HEK-HK␣2b clone 25, and HEK-HK␣2b/ HK␤ g clone 40 cell lines (Fig. 8C). This insensitivity to Sch 28080 was also observed in studies of the full-length HK␣2 subunit expressed in heterologous systems (16,18,19). DISCUSSION Analysis of the regulation of active K ϩ reabsorption in the renal collecting duct and distal colon has been hampered by the lack of structural data concerning potential control mechanisms governing HK␣2 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 ϩ 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 ϩ -K ϩ -ATPase transcript up-regulated in the renal medulla of K ϩdeprived 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 ϩ conservation during chronic hypokalemia, and they uncover a new degree of regulatory complexity for the X ϩ -K ϩ -ATPase ␣ subunit gene family.
The first variant, HK␣2a, is the previously described (17) 1036-amino acid protein, which is encoded by a 4.0-kb mRNA transcribed from the 5Ј-most putative promoter. Primer extension analysis places the major transcription initiation site 274 bp upstream of the translation initiation methionine. Exon 1 includes the 5Ј-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 ϩ -K ϩ -ATPase ␣ subunit family. The analogous exon in ATP1AL1 also encodes 3 amino acids, whereas those for the HK␣1, and Na ϩ -K ϩ -ATPase ␣1 and ␣2 subunits genes encode 4 amino acids, and that for the Na ϩ -K ϩ -ATPase ␣3 subunit gene encodes only 2 amino acids. The 5Ј-flanking region of HK␣2a contains common basal promoter elements. An AT-rich sequence that might serve as a TATA element begins 21 bp 5Ј 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 5Ј-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 5Ј end of the HK␣2 gene, other potential regulatory elements may reside upstream of the sequence we characterized.
The second variant, HK␣2b, has not been previously recognized. This 929-amino acid protein is also encoded by an ϳ4.0-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 ϩ -K ϩ -ATPase ␣ subunit. Primer extension and 5Ј-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 5Ј-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 HK␣2b mRNA is the complex 5Ј-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 Ͻ10% of vertebrate mRNAs (45) and that in some instances they inhibit translational initiation at the major ORF. For example uORFs in the 5Ј-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 86 Rb ϩ 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 ϩ -K ϩ -ATPase ␤ subunit other than the endogenous Na ϩ -K ϩ -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 86 Rb ϩ uptake activity and was required for survival in 1 M 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 ϩ -K ϩ -ATPase ␤1 and HK␤ g subunit support comparable rates of K ϩ uptake (16), it remains to be determined whether the two HK␣2 isoforms differ in their promiscuity for X ϩ -K ϩ -ATPase ␤ subunits.
The functional and regulatory significance of the N-terminal truncation of HK␣2b remains to be explored in further detail. The N terminus is the most variable structural region among the X ϩ -K ϩ -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 ϩ -K ϩ -ATPase exhibited altered K ϩ 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 ϩ -K ϩ -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 IC 50 for ouabain inhibition (400 -800 M in the presence of external 1 mM K ϩ ) 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 IC 50 of 400 -600 M in the presence of external 1 mM K ϩ for HK␣2 pumps expressed in Xenopus oocytes, and Cougnon et al. (18) reported K i values for ouabain of ϳ70 and ϳ970 M in the presence of external 0.2 and 5 mM K ϩ , 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 ϩ -ATPase activities that are distinguished by their kinetic and pharmacological properties in rat kidney (51,52). One activity (type I) is K ϩ -, but not Na ϩ -dependent, ouabain-resistant, Sch 28080sensitive, and expressed in collecting ducts. A second activity (type II) is K ϩ -, but not Na ϩ -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 ϩ depletion. A third activity (type III) is activated by either Na ϩ or K ϩ , 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 ϩ -ATPase activities have been identified in the apical membranes of colonocytes from the distal colon (10), yet only HK␣2 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 ϩ -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 HK␣2 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 ϩ -K ϩ -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 ϩ 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 ϩ 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 ϩ 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 ϩ depletion (presumed to promote secondary hyperaldosteronism), but not chronic K ϩ depletion, enhanced HK␣2 mRNA and protein levels in distal colon. Conversely, chronic K ϩ depletion promoted HK␣2 protein but not mRNA expression in outer medulla, whereas Na ϩ 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 ϩdeprived 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 ϩ 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 HK␣2 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 ϩ -K ϩ -ATPase ␣ subunit isoforms. As additional structure-function and structure-regulation correlations for these genes are identified, their evolutionary relationship should come into clearer focus.