Partial cloning and characterization of Slc12a2: the gene encoding the secretory Na+-K+-2Clminus cotransporter

Jeffrey Randall, Tina Thorne, and Eric Delpire

Renal Division, Department of Medicine, Brigham and Women's Hospital, and Harvard Center for the Study of Kidney Diseases, Harvard Medical School, Boston, Massachusetts 02115

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The Slc12a2 gene encodes a widely expressed bumetanide-sensitive Na+-K+-2Cl- cotransporter that participates in various functions such as Cl- secretion and cell volume regulation. We isolated and characterized 75 kilobases of the murine gene encoding the cotransporter. The cotransport protein is encoded by 27 exons. Ribonuclease protection assay and primer extension demonstrated tissue-specific transcription initiation sites located within 270 base pairs upstream of the start codon. Nucleotide sequence analysis of the proximal 5'-flanking region revealed the presence of a weak TATA box, multiple Sp1/GC consensus sites, and the consensus sequence of a putative transcriptional initiator. Transfection of luciferase reporter gene constructs in mouse inner medullary collecting duct (mIMCD-3) cells confirmed the location of the minimal promoter within a 120-base pair fragment upstream of the cDNA. We also report the identification of an alternatively spliced variant of the cotransporter, expressed primarily in brain. This new spliced variant lacks exon 21, which encodes a 16-amino acid peptide located in the COOH-terminal tail of the protein. The absence of this exon causes the loss of the single protein kinase A consensus site of the cotransport protein.

promoter; spliced variant; brain

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

IN MAMMALS, the bumetanide-sensitive Na+-K+-2Cl- cotransporter is encoded by two distinct genes: Slc12a1 [bumetanide-sensitive Na+-K+-2Cl- cotransporter (BSC1) or NKCC2] and Slc12a2 (BSC2 or NKCC1). The first gene, located on chromosome 2 in the mouse, encodes a kidney-specific Na+-K+-2Cl- cotransporter (5, 17) with a pattern of expression restricted to the luminal (apical) membrane of the thick ascending limb (TAL) and macula densa cells (12). This isoform of the Na+-K+-2Cl- cotransporter in the TAL of Henle participates in NaCl reabsorption. Recently, the exon/intron structure of the human absorptive isoform of the Na+-K+-2Cl- cotransporter was described in a report linking the cotransporter to Bartter's syndrome, a well-known renal salt-wasting disorder (23). The promoter region of the murine absorptive Na+-K+-2Cl- cotransporter gene was recently cloned and partially characterized (8). As expected for a protein that exhibits such a confined pattern of expression, the authors demonstrated a cell-specific activity of the cotransporter promoter. Significant activity was demonstrated in a mouse TAL cell line, whereas no activity was seen in other renal cell lines, such as the S1 proximal tubule cell line, or in nonrenal cell lines, such as NIH 3T3 fibroblasts and HeLa cells.

The second gene, located on chromosome 18 in the mouse, encodes a Na+-K+-2Cl- cotransporter that is broadly expressed (3, 18, 28). In accordance with its broad pattern of expression, this cotransporter serves multiple functions: it participates in Cl- secretion in salivary (2) and airway epithelia (27), acid secretion in the stomach (26), salt and/or acid secretion in the kidney (11), K+ and Cl- homeostasis in the brain (19), and cell volume regulation in a wide variety of cells (16). Although the literature focuses on the regulation of the cotransporter at the protein level, mainly through phosphorylation (for review see Ref. 10), emergence of molecular tools will allow further study of the cotransporter regulation at the gene level. The characteristics of the gene encoding this transporter are unknown. There is also no reported evidence that the gene produces alternative spliced variants. The purpose of the present investigation was to isolate and characterize the gene encoding the mouse secretory isoform of the Na+-K+-2Cl- cotransporter (mBSC2/KCC1) and to identify alternative spliced variants of this gene. We describe the isolation of the Slc12a2 gene, identify its promoter region, and also report the identification of an alternatively spliced variant of the cotransporter that is abundantly expressed in the brain and lacks the single protein kinase A (PKA) consensus site of the previously reported mBSC2 protein.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Mouse genomic library. Two mouse genomic libraries (L129 and Balb/c) were kindly provided by Dr. Richard Maas (Howard Hughes Medical Institute, Dept. of Genetics, Brigham and Women's Hospital). Both libraries had been prepared from a Sau3A I partial digestion of genomic DNA, and the size-selected fragments were inserted into EMBL3. Several million clones were screened using the following protocol: competent LE392 cells were infected with the phage for 20 min at 37°C and plated on top agarose in 150-mm agar plates to obtain 20,000 plaque-forming units per plate. Plaques were lifted onto nylon transfer membrane (NEN-Dupont, Boston, MA), denatured with 0.4 N NaOH and 1.5 M NaCl for 30 min, neutralized with 0.5 M tris(hydroxymethyl)aminomethane (Tris) for 15 min, and washed with 2× SSC for 5 min (1× SSC = 150 mM NaCl and 15 mM sodium citrate). The membranes were then ultraviolet cross linked and hybridized at 42°C for 16 h with 32P-labeled cDNA probes made from different regions of the mBSC2 cDNA. After secondary screening, single positive clones were grown in liquid culture, and the phage DNA was isolated and mapped with a panel of restriction enzymes. Southern blot analysis was performed to identify exon-containing fragments. By use of appropriate restriction enzymes, the phage inserts were digested into shorter fragments and subcloned into pBluescript SK(+) [pBSK(+)]. Exons were sequenced by the dideoxynucleotide termination method with Sequenase DNA polymerase (version 2.0, US Biochemical) using flanking primers and Na+-K+-2Cl- cotransporter-specific primers. The reactions were run on 6% polyacrylamide-urea gels. Sequence analyses were performed using Geneworks 2.2 (IntelliGenetics, Mountain View, CA).

5'-Primer extension. Total RNA from brain and salivary gland (10 µg) was incubated with 500 pmol of the following 5'-reverse primer: TAgAgAgAgAAAgAgTCACAgCg. After annealing, reverse transcription was performed in the presence of 50 mM Tris · HCl, 75 mM KCl, 3 mM MgCl2, 20 mM dithiothreitol, 500 µM dGTP/dATP/dTTP, 40 µCi of [32P]dCTP, and 400 U of SuperScript reverse transcriptase (GIBCO BRL) for 2 h at 37°C. The products were purified by phenol-chloroform extraction and precipitation, resuspended in 10 µl of buffer (48% formamide, 10 mM EDTA, 0.025% bromphenol blue, 0.025% xylene C FF), diluted 100-fold in the same buffer, denatured at 95°C for 5 min, and loaded on 6% polyacrylamide-urea gel. Radiolabeled RNA ladder was loaded on an adjacent lane to size the extended products.

Ribonuclease protection assay. For mapping the start of transcription, a 240-base pair (bp) (Nar I-BsiW I) fragment in pBSK(+) was linearized with Sal I, and antisense riboprobe was made using T3 RNA polymerase and [alpha -32P]UTP. Hybridization of 1 × 105 cpm probe with 10 µg of total RNA from yeast, brain, and salivary gland was performed overnight at 50°C in a solution containing 400 mM NaCl, 1 mM EDTA, 40 mM piperazine-N,N'-bis(2-ethanesulfonic acid), pH 6.4, and 50% formamide. The samples were then treated with a 1:100 dilution of ribonuclease (RNase) A (250 U/ml) and RNase T1 (10,000 U/ml) for 30 min at 30°C. After ethanol precipitation and lyophilization, the pellet was resuspended in loading buffer, denatured at 95°C for 5 min, and separated on a denaturing 6% polyacrylamide gel. For the alternatively spliced transcript, a 731-bp EcoR V fragment from the 4.7-kilobase (kb) mBSC2 cDNA (bp 2632-3363) was subcloned into the EcoR V restriction site of pBSK(+). After linearization of the vector with Sal I, an antisense riboprobe was made using T3 RNA polymerase and [alpha -32P]UTP. Probe (1 × 105 cpm) was hybridized with 30 µg of total RNA from yeast and brain and treated as described above. To accurately determine the sizes of the protected fragments, the isotopic DNA sequencing size marker SequaMark (Research Genetics, Huntsville, AL) was loaded on an adjacent lane.

Promoter activity. Different DNA fragments from the 5'-flanking region of the Slc12a2 gene were ligated upstream of the promoterless luciferase pGL3 basic vector (Promega). Subconfluent (80-90%) 60-mm dishes of mouse inner medullary collecting duct (mIMCD-3) cells (20) were washed twice with Opti-Mem medium (GIBCO BRL) and incubated for 6 h with 1.5 ml of transfection mixture containing 1.5 µg cnv-beta -galactosidase vector (Promega), 2.5 µg of pGL3 construct, and 10 µg/ml polybrene (American Bioanalytical) in Opti-Mem. The transfection mixture was then removed, and the cells were exposed for 3 min to 30% dimethyl sulfoxide in Opti-Mem, then washed rapidly three times with Opti-Mem and incubated for 36 h in complete medium (Dulbecco's modified Eagle's medium-F-12 complemented with 10% fetal calf serum). Then the cells were washed twice with phosphate-buffered saline and lysed with 300 µl of lysis buffer (Promega). beta -Galactosidase activity was obtained by incubating 100 µl of lysate to 100 µl of 2× ONPG buffer (120 mM Na2HPO4, 80 mM NaH2PO4, 2 mM MgCl2, 100 mM beta -mercaptoethanol, and 4.4 mM o-nitrophenyl-beta -D-galactopyranoside) at 37°C for 24 h, adding 334 µl of 1 M Na2CO3, and measuring optical density at 420 nm. Luciferase activity was measured in an EG & G Autolumat luminometer (model LB953) by injecting 100 µl of luciferase reagent (Promega) into 40 µl of lysate. Results were expressed as ratio of luciferase to beta -galactosidase activity. beta -Galactosidase activity was determined to control for changes in transfection efficiency.

RNA isolation. Mouse tissues were promptly isolated and frozen into liquid nitrogen. One gram of tissue was homogenized in 7 ml of a solution containing 4 M guanidine isothiocyanate, 22 mM Na-acetate, and 1.12 g/ml beta -mercaptoethanol. The homogenates were then layered on 4 ml of a 5.7 M CsCl-24 mM Na-acetate solution in Polyallomer ultracentrifuge tubes (Beckman). The tubes were spun overnight at 32,000 rpm at 20°C. The supernatant was then discarded, and the RNA pellet was resuspended in water and concentrated by ethanol precipitation. Finally, the RNA samples were resuspended into diethyl pyrocarbonate-treated water and quantitated by spectrophotometry at 260 and 280 nm.

Reverse transcriptase polymerase chain reaction. Reverse transcription was performed using 2 µg of total RNA in a reaction containing 50 mM KCl, 10 mM Tris · HCl, pH 9.0, 0.1% Triton X-100, 2.5 mM MgCl2, 10 mM dithiothreitol, 1 mM each dNTP, 19 ng/µl random hexamers, 20 U of RNAsin (Promega), and 200 U of the Moloney murine leukemia virus reverse transcriptase. The RNA was denatured at 65°C for 10 min, and the reverse transcription was performed for 1 h at 37°C followed by 10 min of denaturation at 95°C. Polymerase chain reaction (PCR) was then performed using 1 µl of reverse-transcribed DNA in a reaction containing 50 mM KCl, 10 mM Tris · HCl, pH 9.0, 0.1% Triton X-100, 2 mM MgCl2, 200 µM each dNTP, 0.4 µl of [alpha -35S]dATP, 250 ng of primers, and 1.25 U of Taq polymerase (Perkin Elmer). A total of 14 primer sets was used to amplify most of the 4.7-kb mBSC2 cDNA (Table 1). With the exception of the first and last exons as well as the large exon 27, all exons were amplified.

                              
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Table 1.   Sets of PCR primers used for identification of alternative exons

Nondenaturing gels. PCR reactions were separated on nondenaturing 6% polyacrylamide (38% acrylamide-2% bisacrylamide) gels. Samples (10 µl) were denatured for 5 min at 95°C and run at 4°C at 250-300 V overnight with 1× TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA). The gels were then transferred to filter paper, vacuum dried, and exposed to autoradiography for 2-24 h. Selected DNA fragments were excised from the gels with a razor blade, placed in 500 µl of water, boiled for 1 h, precipitated with ethanol, lyophilized, and reconstituted in water for PCR reamplification.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Cloning of the Slc12a2 gene. Two mouse (L129 and Balb/c) genomic libraries were screened with different restriction fragments spanning the entire length of the 4.7-kb cDNA clone encoding the mouse "secretory" Na+-K+-2Cl- cotransporter. Nine clones were isolated from the L129 library (clones 2 = 4, 1 = 3 = 6 = C, 7 = A = B). The second library (Balb/c) provided the following single clones: 51, M2, E4, B3, and 31. Restriction mapping, Southern blots, subcloning, and partial DNA sequencing of these genomic clones allowed us to reconstruct most of the gene (Fig. 1). The mBSC2 4.7-kb cDNA is encoded by a large, >75-kb, genomic fragment. As indicated in Table 2, the 4.7-kb cDNA is composed of 28 exons with an average size of 111 bp, when exons 1 and 27, which were eight to nine times larger, were excluded. All but two intron-exon boundaries were sequenced and compared with the consensus sequences for 5'- and 3'-splice sites. The number of bases divergent from the consensus sites for all exon-intron boundaries was very low, giving an overall 91% conservation for the acceptor (CT)(CT)N(CT)AG(AG) consensus site and 81% conservation for the donor (CA)AGGT(AG)AGT consensus site (Table 2).


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Fig. 1.   Structure of Slc12a2 gene. A: screening 2 mouse genomic libraries provided 14 overlapping clones. Note gap between clones E4 and M2. B: 75-kb genomic fragment showing structure of reconstituted Slc12a2 gene. X and B positioning restriction sites are Xba I and BamH I, respectively. Exons 1-27 are numbered. C: exon structure of mBSC2 4.7-kb cDNA. Protein topology is drawn in black, with core protein (transmembrane section) encoded by exons 2-14. Single protein kinase A consensus site of mBSC2 protein (triangle ) is located at boundary between exons 21 and 22, site of alternative splicing in brain (see Figs. 5-7).

                              
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Table 2.   Description of the exon/intron structure of the Slc12a2 gene

Sequence of the 5'-flanking region of the mouse Slc12a2 gene. Figure 2 shows the sequence of an 840-bp genomic fragment overlapping 133 bp of the mBSC2 cDNA. Analysis of the 5'-flanking region reveals 1) a heavily GC-rich content (83%) within the immediate 300 nucleotides upstream of the cDNA sequence, 2) numerous SP1 consensus sites (GGGCGG) and GC boxes, and 3) a 16-bp sequence showing high homology to the transcriptional initiator found in the terminal transferase gene (9). Consistent with this being a putative initiator site, a weak TATA box (TTTAAA) is found 27 bp upstream (9).


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Fig. 2.   Nucleotide sequence of 5'-flanking region of mouse Slc12a2 gene. Nucleotide positions are relative to ATG start of mBSC2 protein (+1) (Ref. 3). Note presence of a weak TATA box (TTTAAA), a putative initiator site (Inr), and multiple Sp1/GC boxes, as well as MEF2, OTF/1-2A, CACCC-binding, NFkappa B, and AP-2 sites. Restriction sites such as Sac I, Bgl II, Nar I, Eag I, and BsiW I that were used for luciferase constructs (see Fig. 4) are located within sequence. Start sites mapped by ribonuclease protection assay and 5'-primer extension in salivary gland and brain are indicated at positions -266 and -194, respectively. Oligonucleotide primer used for 5' extension is also shown at position -128-106.

Mapping the start of transcription. Transcription start sites were mapped in brain and salivary gland using primer extension analysis and RNase protection assay. Brain and salivary gland RNA samples were used, since both tissues express high levels of Na+-K+-2Cl- cotransporter. Primer extension was performed using 500 pmol of an antisense oligonucleotide primer located 25 bp downstream of the 5' end of the cDNA and 30 µg of total RNA. As indicated in Fig. 3A, different-sized products were observed in brain and salivary gland. In the brain an intense band was observed at 89 bp, placing the start site 64 bp upstream of the 5' end of the mBSC2 cDNA. In contrast, a larger product of 159 bp was observed in salivary gland, placing the start site 70 nucleotides further upstream (see sequence in Fig. 2). To confirm the position of these start sites, RNase protection assay was performed using a 32P-labeled antisense riboprobe synthesized from a 240-bp Nar I-BsiW I genomic fragment (see sequence in Fig. 2). As indicated in Fig. 3B, different-sized fragments were protected in brain (155-160 bp) and salivary gland (210 bp), whereas no signal was detected with negative control yeast tRNA. The start sites provided by RNase protection assay matched (±10 nucleotides) those obtained by 5'-primer extension.


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Fig. 3.   Mapping of Slc12a2 start site(s) by 5'-primer extension and ribonuclease (RNase) protection assay. Autoradiograph of a 6% denaturing polyacrylamide gel shows radiolabeled fragments extended (5'-primer extension) or protected (RNase protection) from brain and salivary gland RNA. A: 23-bp oligonucleotide primer (see sequence in Fig. 2) was used for reverse transcription of brain and salivary gland RNA (10 µg) in presence of [32P]dCTP. Note extension of different-sized fragments in brain and salivary gland. B: RNase protection analysis of RNA isolated from brain and salivary gland. RNA (10 µg) was hybridized overnight with 1 × 105 cpm 32P-labeled Nar I-BsiW I probe and digested for 30 min at 30°C with a 1:100 dilution of RNase A + RNase T1 mixture (250 and 10,000 U/ml, respectively). RNase digestion yielded protected fragments of different sizes in brain and salivary gland. Aliquot of free 32P-labeled probe was run in an adjacent lane. A radiolabeled RNA ladder (middle) was loaded on polyacrylamide gel to size extended and protected fragments.

Functional expression of the Slc12a2 gene promoter. To determine whether the 5'-flanking region of the Slc12a2 gene contains a functional gene promoter, a 2,063-bp Hind III-BsiW I fragment containing 79 bp of 5'-untranslated region of the mBSC2 cDNA was ligated upstream of the luciferase gene. mIMCD-3 cells (20) expressing the Na+-K+-2Cl- cotransporter (3) were transfected with the construct as well as with the basic promoterless vector as a negative control. As indicated in Fig. 4, ligation of the 2-kb fragment upstream of the luciferase gene yielded significant luciferase activity compared with the promoterless vector. Successive deletions of the 2-kb construct were made and assayed for luciferase activity (Fig. 4). Deletion of 1,361 bp resulted in increased luciferase activity, suggesting the presence of silencer sequences located within the Hind III-Sac I fragment. Whereas the deletion of 186 bp (Sac I-Bgl II fragment) did not affect significantly the luciferase activity, deletion of a larger 280-bp (Bgl II-Nar I) fragment reduced the activity of the promoter. The deletion of an additional 114-bp Nar I-Eag I fragment resulted in the total loss of promoter activity. These results confirm that the 5'-flanking sequence of the mBSC2 cDNA (Fig. 2) is able to promote gene transcription and that the minimal promoter lies within 160 bp of the 5' end of the mBSC2 cDNA.


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Fig. 4.   5'-Flanking region of Slc12a2 gene induces promoter activity in mouse inner medullary collecting duct (mIMCD-3) cells. Relative luciferase activities generated by different-sized constructs of Slc12a2 gene are compared with promoterless PGL3 basic vector. Note increased activity induced by deletion of 1.3-kb Hind III-Sac I fragment. Also note existence of significant promoter activity in 2 deletion mutants pGL3-516 and pGL3-236 and total loss of activity with shorter Eag I-BsiW I luciferase construct. Values are means ± SD of 3 measurements. All constructs yield significantly higher luciferase activity than basic construct (P < 0.01, by analysis of variance), except activity of pGL3-122, which is not significantly different from pGL3 basic (P > 0.8, by analysis of variance). Experiments were repeated twice with similar results.

Identification of an alternatively spliced variant of Slc12a2. To identify alternatively spliced variants of the BSC2 isoform of the Na+-K+-2Cl- cotransporter, we used cDNA reverse transcribed from a variety of mouse tissues, followed by PCR amplification using a set of oligonucleotide primers designed to amplify two to three exons simultaneously (Table 1). The reactions were then separated on nondenaturing polyacrylamide gels to allow identification of PCR fragments of identical length but different composition (see EXPERIMENTAL PROCEDURES). A pattern of three bands is generally observed: the two bands on the top consist of each single strand of DNA, whereas the bottom band represents the renatured double-stranded PCR fragment. As indicated in Fig. 5A, lane 1, a specific pattern of three bands is observed when a set of primers designed to amplify exon 21 and the mBSC2 cDNA is used as control (solid arrowheads). This pattern is also observed with cDNA reverse transcribed from kidney, stomach, brain, skeletal muscle, salivary gland, and heart RNA. An additional pattern of three bands is observed in brain and, to a lesser extent, in skeletal muscle. Whereas double-stranded bands were not observed in other tissues, faint single-stranded bands were detected, suggesting the presence of the isoform at extremely low abundance in these tissues. Sequence analysis of the additional fragments from brain (Fig. 6A) reveals the absence of 48 nucleotides constituting exon 21 (Fig. 1C). As shown in Fig. 6B, the exclusion of this exon leads to the loss of 16 amino acids and the loss of the only protein kinase A consensus site of the mBSC2 protein. Figure 5 also contains the results of an experiment performed with a set of primers corresponding to another part of the mBSC2 cDNA, where all PCR fragments migrated in identical fashion (Fig. 5B).


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Fig. 5.   A: nondenaturing acrylamide gel (single-strand conformation polymorphism) analysis of polymerase chain reaction (PCR) fragments reveals existence of 2 spliced variants. PCR was performed using a primer pair designed to amplify a region of putative intracellular COOH-terminal tail of mBSC2 protein and cDNA reverse transcribed from kidney, stomach, brain, muscle, salivary gland, and heart. mBSC2 4.7-kb cDNA was used as positive control (lane 1). Note presence of 3 bands in mBSC2 lane, corresponding to 2 single strands of PCR fragment (top bands) and renatured double-stranded fragment (bottom band). This migration pattern, corresponding to original mBSC2 isoform, is present in every tissue examined (lanes 2-7). Note additional pattern of 3 bands (open arrowheads) in brain and skeletal muscle. Whereas extremely faint single-stranded fragments can be seen in other tissues, no renatured double-stranded fragments are visible. B: 1 single pattern of migration (3 bands, solid arrowheads) is observed with a set of oligonucleotide primer designed to amplify a fragment spanning 2nd transmembrane domain of mBSC2 protein. Autoradiographs were exposed for 2-3 h.


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Fig. 6.   Comparison of nucleotide sequence of brain PCR fragment with mBSC2 cDNA. A: new fragment observed in brain sample (Fig. 5) was excised from acrylamide gel, PCR reamplified, and subcloned into pBluescript SK(+) for sequencing. Nucleotide positions corresponding to mBSC2 4.7-kb cDNA (Ref. 3) are numbered on right. Boxed residues represent nucleotide identity. Note absence of a 48-nucleotide stretch in brain fragment. B: analysis of sequences reveals loss of exon 21, which encodes for a 16-amino acid peptide and replacement of a basic residue (K, lysine) to a neutral residue (Q, glutamine), leading to loss of a putative cAMP-dependent protein kinase A (PKA) consensus site.

Evidence for the existence of an alternative transcript in the mouse brain. RNase protection assay was used to verify expression of the alternative transcript in the brain. A 731-bp EcoR V fragment from the 4.7-kb mBSC2 cDNA was subcloned into the unique EcoR V site of pBSK(+). Antisense 32P-labeled riboprobe was hybridized with 60 µg of tRNA from yeast and mouse brain and incubated in the presence of RNase. As indicated in Fig. 7, the riboprobe was entirely digested with the yeast RNA sample, whereas three major fragments were protected with brain RNA: a full-length (731-bp) fragment and two smaller fragments of 405 and 277 bp. These two fragments represent the segments between the EcoR V sites and exon 21. This experiment demonstrates unambiguously that two distinct transcripts are present in the brain, one containing and one lacking exon 21. The alternative transcript from brain was cloned by PCR. Two overlapping PCR fragments were generated: the first fragment was obtained using primers designed in exons 1 and 22, whereas the second fragment was generated with primers located in exons 20 and 28. Each PCR fragment was ligated into the vector pCRII (Invitrogen) and transformed in Escherichia coli. The two overlapping PCR fragments were compared with the mBSC2 clone by PCR followed by nondenaturing acrylamide gel electrophoresis, using the primer sets listed in Table 1. It was shown that the brain alternatively spliced variant was otherwise identical to the mBSC2 clone.


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Fig. 7.   RNase protection assay demonstrates existence of 2 transcripts in brain. A 730-bp 32P-labeled antisense cRNA probe was incubated with 30 µg of yeast RNA (lanes 1 and 2) or mouse brain RNA (lanes 3 and 4) and digested with RNase A + RNase T1 at 1:50 (lanes 1 and 3) or 1:100 dilution (lanes 2 and 4). Reactions were separated on denaturing polyacrylamide gel. An aliquot of free probe was loaded on an adjacent lane (lane 5). Note absence of any protected fragment in yeast RNA sample and presence of 3 major protected fragments in brain sample (arrowheads). 730-Nucleotide band (top) represents full-length protected fragment; 405- and 277-nucleotide fragments correspond to the 2 fragments flanking exon 21.

Expression of the alternative spliced variant within the brain. In a previous study we showed abundant expression of the mBSC2 Na+-K+-2Cl- cotransporter in choroid plexus and in neurons throughout the brain (19). In a first attempt to localize the alternative transcript, we performed PCR followed by nondenaturing acrylamide gel electrophoresis. We used cDNA reverse transcribed from choroid plexus, hypothalamus, cerebellum, brain stem, and cortex. Figure 8 demonstrates that the two transcripts are present in all areas of the brain examined, except in the choroid plexus, where the alternatively spliced variant is not expressed.


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Fig. 8.   Nondenaturing polyacrylamide (single-strand conformation polymorphism) gel showing distribution of alternatively spliced variant in brain. Total RNA isolated from choroid plexus, hypothalamus, cerebellum, brain stem, and cerebral cortex was reverse transcribed, and cDNAs were used in PCR reactions with primers designed to amplify region of exon 21. Although original isoform (solid arrowheads) was expressed in all regions of brain, alternatively spliced variant (open arrowheads) is not expressed in choroid plexus. Autoradiograph was exposed for 3 h.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Electroneutral Na+-K+-2Cl- cotransporters participate in a variety of physiological functions. In the TAL, the BSC1 isoform of the cotransporter mediates NaCl reabsorption (5, 17). This absorptive isoform of the cotransporter is found only in the kidney, with a pattern of expression further restricted to medullary and cortical thick limbs, and macula densa cells (12). In contrast, epithelial salt secretion is mediated by a second isoform of the Na+-K+-2Cl- cotransporter (BSC2) encoded by a distinct gene (3, 18, 28). In respiratory mucosa (27), salivary gland (2), lacrimal gland (21), and inner medullary collecting duct (11), the BSC2 cotransporter is located on the basolateral membrane, where it constitutes the entry pathway for Na+ and Cl-. This secretory isoform of the cotransporter participates in other important physiological functions, such as acid secretion in the stomach (26), regulation of cerebrospinal fluid composition (19), Cl- homeostasis in neurons (1, 19), and cell volume regulation in a wide variety of cells (16).

With the goal to study molecular mechanisms of Na+-K+-2Cl- cotransporter gene regulation, we set out to clone and characterize the murine Slc12a2 gene and its promoter region. We used the 4.7-kb mBSC2 cDNA to screen two phage genomic libraries. Fourteen clones were isolated, mapped by restriction digest, and analyzed by Southern blot. Two large genomic fragments spanning 75 kb were reconstituted, comprising most of the gene encoding the 4.7-kb mBSC2 cDNA. The gene was not characterized in its entirety, since a clone overlapping the two large genomic fragments and containing exon 16 was not isolated and the identity of the 2.8-kb extension of the 3'-UTR (3) is still unknown. Whereas the Slc12a2 gene seems to be larger than the human gene encoding for the thiazide-sensitive Na+-Cl- cotransporter (~55 kb) (24), it is about equal to or slightly larger than the human "absorptive" Na+-K+-2Cl- cotransporter (~80 kb) (23). The mBSC2 transcript is encoded by 28 exons (or more if we include the 3'-UTR extension) compared with the two other transporter mRNAs, which are encoded by 26 exons. The introns of the Slc12a2 gene vary from 85 bp (between exons 8 and 9) to >15 kb (between exons 1 and 2).

Previously, we demonstrated by RNase-H mapping that the beginning of the cloned mIMCD-3 mBSC2 cDNA was very close to the start of the mRNA molecule (<100 bp). Our present results indicate that the 5'-flanking region located immediately upstream of exon 1 includes the promoter region of the Slc12a2 gene. The transcription start site(s) of Slc12a2 was mapped using primer extension analysis and RNase protection assays in tissues expressing high amounts of Na+-K+-2Cl- cotransporter, e.g., brain and salivary gland. Both mapping methods provided identical start sites: a single site located 65 bp upstream of the first nucleotide of the 4.7-kb mBSC2 cDNA for brain and a distinct site for salivary gland located 70 bp further upstream. The 5'-flanking region of the Slc12a2 gene was partially sequenced, revealing the presence of a heavily GC-rich (83%) region located immediately upstream of the cDNA. This region includes multiple Sp1/GC boxes as well as a 16-bp nucleotide sequence with high homology to the initiator element from the terminal transferase gene (9) located downstream of a TTTAAA sequence. The Slc12a2 gene promoter is unusual, since it features both characteristics typical for housekeeping genes, i.e., absence of strong TATA and CAAT boxes and presence of multiple binding sites for the Sp1 transcription factor (4, 25), and the characteristics of a more common promoter with the presence of an initiator element located 25-30 bp downstream of a weak TTTAAA sequence. As discussed by Javaheri and co-workers (9), initiator elements can cooperate with a TATA box if both elements are found in the same core promoter and are separated by ~25 bp. Under these conditions, the strength of the promoter is typically dependent on the type and position of the TATA sequence (9). Evidence has been presented showing that not only is there protein interaction between initiator elements and the TATA box but also between initiator elements and upstream Sp1 sites (13). Because the transcription start sites mapped in brain and salivary gland are located upstream of the initiator element, one has to conclude that the TTTAAA and initiator sequences are not involved in promoting transcription in these two tissues. Unlike other constitutively expressed genes that have housekeeping functions, the initiation of transcription occurred at one major site rather than multiple sites. This unusual feature has also been reported in other genes, e.g., the nerve growth factor receptor gene (22). Binding sites for different transcription factors such as MEF2, CACCC binding, OTF/1-2A, NFkappa B, and AP-2 were also identified. Their potential role in regulating Slc12a2 transcription will need to be established.

To verify that the 5'-flanking region of the Slc12a2 gene contained a functional gene promoter, we ligated a 2,063-bp Hind III-BsiW I fragment to the pGL3 promoterless luciferase gene and expressed it in mIMCD-3 cells. This fragment was competent to initiate transcription when cloned upstream of the heterologous reporter gene. Expression of a shorter construct (700-bp Sac I-BsiW I fragment) produced significantly higher promoter activity, suggesting the presence of silencer element(s) within the upstream 1,361-bp fragment. To narrow down the location of the minimal promoter, we also examined the activity of additional 5'-deletion mutants. Whereas promoter activity was not significantly affected when 186 bp were deleted, deletion of 280 bp resulted in a reduction of promoter activity, suggesting the existence of enhancer(s) elements within the Bgl II-Nar I fragment. Complete loss of promoter activity was demonstrated after deletion of a 122-bp Eag I-BsiW I fragment, thus confirming the location of the minimal promoter of the Slc12a2 gene between the Nar I and Eag I restriction sites (see sequence in Fig. 2).

Finally, using a PCR approach, we set out to identify alternatively spliced products of the Slc12a2 gene. The PCR products were analyzed by single-strand conformation polymorphism (SSCP) to distinguish fragments of identical length but different composition. We used 14 sets of primers (Table 1), spanning most of the exons encoding the 4.7-kb mBSC2 cDNA. Alternative splicing of the first, last, and large exon 27 were not considered in the analysis. We identified an alternatively spliced variant of the cotransporter that was mostly expressed in the brain and, to a lesser extent, in skeletal muscle. The existence of two brain transcripts was verified by RNase protection assay. Sequence analysis of the new variant revealed that it lacked exon 21 but was otherwise identical to the original protein.

In a previous study we demonstrated expression of the BSC2 cotransporter in choroid plexus and neurons (19). Although no staining was observed in glial cells and endothelial cells, we did not rule out the possibility of cotransporter expression in these cells below the limit of detection of our immunohistochemistry method. The demonstration of alternative splicing of the cotransporter in the brain within the region recognized by our polyclonal antibodies (amino acids 938-1011) raises the intriguing possibility that the antibodies do not recognize the alternatively spliced variant. To address this issue, we created a fusion protein corresponding to the alternatively spliced variant. While lacking the 16 amino acids encoded by exon 21, the fusion protein retained the same 58 amino acids flanking the alternatively spliced exon. Western blot analysis revealed that the purified antibodies recognize both variants of the cotransporter. Whether this result can be extended to immunolocalization remains to be demonstrated. Using in situ hybridization with oligonucleotide probes designed within and without exon 21, we attempted to localize this new variant of the cotransporter within the brain. No specific probe could be used to identify the alternatively spliced variant. Both probes showed identical staining of choroid plexus and in neuron-rich regions, so no differences in expression pattern were observed (not shown). We then relied on the SSCP method and demonstrated that the choroid plexus did not express the new variant (Fig. 8). Although we cannot rule out expression of the alternatively spliced variant in vasculature or glial cells, it is likely that neurons coexpress the two alternatively spliced variants of the BSC2 cotransporter. This hypothesis would explain the identical in situ hybridization patterns observed with the two oligonucleotide probes. Expression of the new variant of the cotransporter in neurons could also account for the extremely low levels of expression detected in other tissues by the very sensitive PCR method. Indeed, observation of low expression of the alternatively spliced variant in kidney, stomach, salivary gland, and heart may be due to neuronal (nerve) contamination of dissected tissues.

Exon 21 encodes for a 16-amino acid peptide located in the COOH-terminal tail of the cotransporter protein. The physiological significance of this exon is unknown. It is tempting to speculate, however, that this region of the protein participates in the regulation of the cotransporter, since the absence of exon 21 results in the loss of the only PKA consensus site of the BSC2 cotransporter. The role of PKA in Na+-K+-2Cl- cotransport has been investigated in different systems. In many cell types, activation of cAMP-dependent PKA leads to increased activity of the cotransporter (for reviews see Refs. 6 and 10). In certain tissues, e.g., vascular endothelial cells, cAMP elevation does not lead to increased Na+-K+-2Cl- cotransport activity (15). Whether expression of this alternatively spliced variant accounts for such differences in cAMP responsiveness remains to be determined. Expression of both variants in cells deficient of cotransport activity should uncover the physiological role of this short COOH-terminal segment of the cotransporter protein.

Although we cannot rule out the existence of other spliced variants, especially within the 3' end of the mBSC2 protein, we have covered with our PCR/SSCP analysis most of the cotransport protein. Considering its very broad pattern of expression, it is intriguing that only one alternatively spliced variant of the cotransporter was found. This observation contrasts to the multiplicity of spliced variants generated by the Slc12a1 (BSC1) gene, which encodes the absorptive Na+-K+-2Cl- cotransporter and has a pattern of expression that is restricted to a specific region of the kidney. Six distinct spliced variants of the BSC1 protein have been identified: it was first demonstrated that exon 4 in rabbit (17), mouse (7), and human (23) exists in three different variants (exons 4A, 4B, and 4F). It was then shown that each of these variants exists with two alternative 3' termini, giving rise to six distinct spliced variants (14). The fourth exons of the BSC1 and BSC2 proteins are homologous and have identical length, but this exon in BSC2 is not alternatively spliced (Fig. 5B).

    ACKNOWLEDGEMENTS

This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-49251, American Heart Association (National Center) Grant-in-Aid 96007060, and a grant from the Polycystic Kidney Research Foundation. E. Delpire is an Established Investigator of the American Heart Association.

    FOOTNOTES

Address for reprint requests: E. Delpire, Laboratory of Cellular and Molecular Physiology, Dept. of Anesthesiology, Vanderbilt University School of Medicine, 1313 21st Ave. South, 504 Oxford House, Nashville, TN 37232-2372.

Received 25 March 1997; accepted in final form 12 June 1997.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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