Isoforms of the Na-K-2Cl cotransporter in murine TAL I. Molecular characterization and intrarenal localization

David B. Mount1, Allan Baekgaard2, Amy E. Hall1, Consuelo Plata3, Jason Xu1, David R. Beier4, Gerardo Gamba3, and Steven C. Hebert1

1 Division of Nephrology, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee 37232; 4 Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115; and 3 Molecular Physiology Unit, Department of Nephrology and Mineral Metabolism, Instituto Nacional de la Nutrición Salvador Zubirán and Instituto de Investigaciones Biomédicas, National University of Mexico, Mexico City CP 14000, Mexico; 2 Department of Medical Physiology, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have identified several alternatively spliced cDNAs encoding mBSC1, an apical bumetanide-sensitive Na+-K+-2Cl- cotransporter from mouse kidney. Two full-length clones were isolated, designated C4 and C9, predicting proteins of 770 and 1,095 amino acids, respectively. The C4 isoforms are generated by utilization of an alternative polyadenylation site located within the intron between exons 16 and 17 of the mBSC1 gene on chromosome 2; the resultant transcripts predict a truncated COOH terminus ending in a unique 55 amino acid sequence. The predicted C4 and C9 COOH termini differ in the distribution of putative phosphorylation sites for both protein kinase A and C. Independent splicing events involve three previously described cassette exons, which are predicted to encode most of the second transmembrane domain. A total of six different isoforms are expressed, generated by the combinatorial association of three cassette exons and two alternative 3' ends. C9-specific and C4-specific antibodies detect proteins of ~150 and 120 kDa, respectively, in mouse kidney. Immunofluorescence and immunohistochemistry indicate expression of both COOH-terminal isoforms within the thick ascending limb of the loop of Henle (TAL). However, staining with the C4 antibody is more heterogeneous, with a decreased proportion of positive cells in the cortical TAL. Functional expression in Xenopus oocytes indicates a dominant negative function for C4 isoforms [companion study, C. Plata, D. B. Mount, V. Rubio, S. C. Hebert, and G. Gamba. Am. J. Physiol. 276 (Renal Physiol. 45): F347-F358, 1999], and the differential expression of these isoforms may contribute to functional heterogeneity of Na+-K+-2Cl- cotransport in mouse TAL.

sodium-potassium-chloride cotransporter; bumetanide; protein kinase A; adenosine 3',5'-cyclic monophosphate; thick ascending limb of Henle; alternative splicing


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ELECTRONEUTRAL cation-chloride cotransporters have been described in a variety of epithelial and nonepithelial cells. These transporters were initially separated on the basis of differential sensitivity to loop diuretics and thiazide diuretics; however, recent molecular efforts have demonstrated that they form a family of structurally homologous proteins (21). Thus far, five gene products have been identified in vertebrates, encoding an apically expressed bumetanide-sensitive Na+-K+-2Cl- cotransporter (10, 14, 24, 29, 33), a predominantly basolateral bumetanide-sensitive Na+-K+-2Cl- cotransporter (7, 21), an apical thiazide-sensitive Na+-Cl- cotransporter (10, 11), a widely expressed K+-Cl- cotransporter, and a neuronal K+-Cl- cotransporter (21).

In the renal thick ascending limb of the loop of Henle (TAL), apical bumetanide-sensitive Na+-K+-2Cl- transport plays a critical role in the countercurrent mechanism, maintenance of the extracellular fluid volume, and renal reabsorption of calcium and magnesium. We previously identified a cDNA encoding the apical Na+-K+-2Cl- cotransporter rBSC1 from the outer medulla of rat kidney and demonstrated bumetanide-sensitive Na+- and Cl--dependent uptake of rubidium (86Rb+) in Xenopus laevis oocytes injected with rBSC1 cRNA (10). Orthologous cDNAs, designated NKCC2 by Forbush and colleagues have also been isolated from rabbit (24), mouse (14), and human kidney (29, 33). The BSC1 gene is expressed exclusively within the kidney, and the rBSC1 protein has been localized to the apical membrane of epithelial cells in both the medullary and cortical TAL (MTAL and CTAL, respectively) (8, 16). Mutations in the human BSC1 gene have been demonstrated in kindreds with Bartter's syndrome type I, a familial form of hypokalemic metabolic alkalosis (29, 33).

Previous data were suggestive of molecular heterogeneity in the apical bumetanide-sensitive Na+-K+-2Cl- cotransporters of the mouse TAL. Alternative mRNA splicing of BSC1 has been described in several species, affecting three mutually exclusive exon cassettes, different versions of exon 4 (29), near the 5' end of partial cDNA clones (14, 24, 33). Northern blot analysis also indicates that the outer medulla of mouse kidney contains two transcripts of different length, 3.0 and 4.6 kb, which hybridize to the flounder thiazide-sensitive Na+-Cl- cotransporter (flTSC) cDNA (10). Finally, bumetanide binding and photolabeling experiments have detected two immunologically related bumetanide binding proteins in mouse outer medulla, a high-affinity 150-kDa bumetanide binding protein and a low-affinity bumetanide binding protein of ~75 kDa (12).

There is also evidence for functional heterogeneity of apical Na+-K+-2Cl- cotransport in the TAL. We have demonstrated that vasopressin, which stimulates Na+-Cl- transport in the murine MTAL (20), switches cotransport from a completely K+-independent Na+-Cl- mode to a K+-dependent Na+-K+-2Cl- mode (31). The molecular mechanism of this modulation is unknown; however, the recycling of K+ through the K+-dependent Na+-K+-2Cl- cotransporter and apical K+ channels is required for the generation of a lumen-positive potential difference, which drives paracellular absorption of Na+ and other cations (21, 31). Heterogeneity in the response to vasopressin has also been shown in CTAL, where vasopressin has little effect on transepithelial Na+-Cl- transport (13).

To begin the characterization of this functional and molecular diversity, we undertook cloning of the mouse homolog of the renal apical bumetanide-sensitive Na+-K+-2Cl- cotransporter, hereafter referred to as mBSC1. We now report the isolation of cDNA clones derived from alternative splicing of the three cassette exons (14) and the use of two alternative 3' ends and show that this alternative splicing results in a total of six different transcripts. One of the alternative 3' ends predicts a new subtype of mBSC1 isoforms with a COOH-terminal truncation. We also examine the expression and localization of proteins with the two alternative COOH-terminal domains. Functional expression of full-length isoforms is detailed in the companion study (25).


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

cDNA cloning and sequencing. To isolate mBSC1 clones, a directional size-selected (3-6 kb) mouse outer medulla cDNA library was constructed in pSPORT1 (SuperScript, BRL). Poly(A)+ RNA for this purpose was isolated from adult male CD-1 mice by the guanidine isothiocyanate method, followed by selection on an oligo(dT)-cellulose column (type 7, Pharmacia). Colonies were screened with a 32P-labeled randomly primed BamH I-BamH I fragment (nucleotides 1194-2543) of flTSC (11), at moderate stringency. Seventeen clones were isolated, and cDNA inserts were categorized on the basis of size, restriction mapping, and sequencing of 5' and 3' ends. The cDNA inserts of plasmids were sequenced using the Sequenase version 2.0 kit (US Biochemical) and by automated cycle sequencing on an Applied Biosystems model 373A sequencer (Howard Hughes Medical Institute, Harvard Medical School). Analysis of nucleotide and amino acid sequence was performed using the GENEWORKS software package (Intelligenetics, version 2.3.1) and the program pBLAST (2). The nomenclature of the mBSC1 cDNAs is as follows: the letters A, B, or F refer to the cassette exons (14), and numerals 4 and 9 refer to the C4 and C9 alternative 3' ends (see RESULTS).

Northern blots and RT-PCR. RNA was extracted from mouse tissues (CD-1 and C57BL/6J strains), and poly(A)+ RNA was selected from renal cortex and inner and outer stripe of outer medulla using oligo(dT)-resin (Oligotex, Qiagen). RNA [2.5 µg/lane of poly(A)+ RNA and 10 µg/lane of total RNA] was resolved by electrophoresis (5% formaldehyde, 1% agarose), transferred to a nylon membrane (Stratagene), and probed with 32P-labeled randomly primed probes. Hybridization was performed at 50°C in 4× SSCP, 40% formamide, 4× Denhardt's solution, 0.5% SDS, and 200 µg salmon sperm DNA, and membranes were washed twice for 20 min at room temperature in 2× SSCP and 0.1% SDS, and twice for 1 h at 65°C in 0.5× SSCP and 0.1% SDS.

Subclones and restriction fragments of C4 and C9 were generated for use as probes. A 365-bp Hind III-Apa I fragment (nucleotides 1284-1649 of C9.0, 1306-1671 of C4.0) was used as a C4/C9-common probe, and a 475-bp Sac I-Sac I fragment (nucleotides 3293-3768 of C9.0) was used as a C9-specific probe. A 382-bp C4-specific fragment (nucleotides 2530-2912) was generated by PCR and subcloned into pBluescript. High-stringency Southern blot analysis with both the mBSC1 clones and mBSC2 (7) was performed with the individual probes to verify their specificity.

RT-PCR of kidney RNA was performed with C4- and C9-specific primer pairs, which were designed with sense primers within common sequences and antisense primers within the unique 3' ends. The C4-specific primer pair used was
sense, S1: 5′-GACAACGCTCTGGAATTAACC-3′
antisense, AS1: 5′-GTCTGCTTCCTTCTCGTTGG-3′
The amplified product corresponds to nucleotides 2182-2692 of C4.0 (510 bp). The C9-specific primer pair used was
sense, S2: 5′-GTGCATTGTCTTAACAGGCG-3′
antisense, AS2: 5′-GTGTTTGGCTTCATTCTCCC-3′
The amplified product corresponds to nucleotides 2216-2494 of C9.0 (278 bp). Total RNA (200 ng/PCR reaction) from mouse kidney was reverse transcribed using oligo(dT) priming. PCR amplification was performed using Taq polymerase (Stratagene) in a PTC-100 programmable thermal controller (MJ Research), using 30 cycles of denaturation (92°C, 2 min), annealing (54°C, 1 min), and extension (72°C, 1 min). The buffer conditions for primer pairs were optimized using cDNA templates and the Opti-Prime buffer system (Stratagene), which varies Mg2+ and K+ concentration and pH. PCR for the S1/AS1 primer pair was optimal using buffer 5 (10 mM Tris, pH 8.8, 1.5 mM MgCl2, and 25 mM KCl) and 5% formamide. PCR for the S2/AS2 primer pair was optimal using buffer 10 (10 mM Tris, pH 9.2, 1.5 mM MgCl2, and 75 mM KCl). PCR products from both a C4-specific and a C9-specific RT-PCR sample were subcloned into the EcoR V site of pBluescript by blunt-end ligation.

The murine B cassette exon was not identified in any of the cDNA clones obtained above (see RESULTS). Therefore, it was cloned from renal cortex by RT-PCR, using a primer pair that amplifies the equivalent of nucleotides 746-996 of C9.0. PCR products were subcloned into the EcoR V site of pBluescript and screened by PCR with a sense primer (primer S3, defined below) specific for the A cassette; three clones that did not contain the A cassette were sequenced on both strands.

To determine whether alternative splicing of the three 5' cassette exons occurred independently of the splicing that generated the C4 and C9 3' ends, RT-PCR was also performed with sense primers within the cassettes and antisense primers within the unique 3' ends. RT-PCR was performed as above, except that PCR reactions included Taq Extender (Stratagene), RT-treated poly(A)+ RNA (20 ng/PCR reaction) was used as the template, and extension times were increased to 1.5 min. The cassette-specific sense primers were
S3; A cassette-specific 5′-CATGGTAACCTCTATCACGGG-3′
S4; B cassette-specific 5′-GTGTGATTATCATCGGCTTAGC-3′
S5; F cassette-specific 5′-ATCATCATTGGCCTGAGCG-3′
The AS2 primer served as the C9-specific antisense primer; PCR with this primer and the cassette-specific primers generated products of ~1.7 kb. To shorten the amplified products, a primer more 5' than AS1 was used as the C4-specific antisense primer
AS3; C4-specific 5′-CCATGGGTATCTTCTGTGCC-3′
PCR with the cassette primers and the AS3 primer generated products of ~1.6 kb. For the four isoforms not isolated in the original cDNA clones (see RESULTS, Fig. 2A), buffer conditions for PCR were optimized with cDNA templates that had been generated for functional expression (see Ref. 25). PCR for the S4/AS3 primer pair was optimal in Stratagene buffer 5 (see above). Buffer 9 (10 mM Tris, pH 9.2, 1.5 mM MgCl2, and 25 mM KCl) was used for the S5/AS3 primer pair. PCR for the S3/AS2 primer pair utilized buffer 10 (see above), and buffer 9 was used for the primer pair S4/AS2. The PCR adjunct 5% formamide was also added for the S4/AS3, S5/AS3, and S4/AS2 primer pairs. Amplified RT-PCR bands of the appropriate size were subcloned into the EcoR V site of pBluescript.

Characterization of mBSC1 proteins. A quantity of 1.0 µg of linearized cDNA (mBSC1-A4 and mBSC1-F9) was translated in vitro using T7 RNA polymerase-coupled rabbit reticulocyte lysate (TNT T7, Promega) and [35S]methionine for 1 h at 30°C. Protein was resolved by SDS-PAGE (10% polyacrylamide), along with prestained high and low range SDS-PAGE molecular weight standards (Bio-Rad, Hercules, CA).

Western blotting of outer medulla membrane protein was performed using antibodies specific for both alternatively spliced mBSC1 COOH termini. A previously characterized antibody generated against a COOH-terminal segment of rBSC1 (amino acids 834-900) was used as a C9-specific antibody (16); these amino acids are not present in the predicted C4 protein. This region of mBSC1 is 94% homologous to rBSC1, and the specificity of the antibody has been established in rat (16). A C4-specific antibody was also generated (Quality Control Biochemicals, Hopkinton, MA) by immunization of rabbits with the HPLC-purified peptide acetyl-CGTEDTHGNRKEKKR (amino acids 735-749 of mBSC1-A4, with an amino terminal cysteine). This peptide lies within the unique COOH terminus of mBSC1 C4 isoforms and does not display any homology to any other proteins in a BLAST search (2) of the nonredundant protein database. For isolation of membrane protein, mouse kidney (CD-1 and C57BL/6J strains) was dissected, homogenized in 9 vol of ice-cold homogenization buffer (0.32 M sucrose, 5 mM Tris, and 2 mM EDTA, pH 7.5, plus protease inhibitors), and centrifuged at 3,000 g for 10 min. The supernatant was subsequently centrifuged at 100,000 g at 4°C for 30 min, followed by resuspension of the pellet in homogenization buffer. Membrane protein (100-150 µg) was resolved by gel electrophoresis in 6% SDS-polyacrylamide, and transferred in 25 mM Tris · HCl, 192 mM glycine, and 25% methanol to a polyvinylidene difluoride membrane. Affinity purification of antibody and Western blotting was performed as described (34). Antigen-antibody complexes were visualized using enhanced chemiluminescence (ECL system, Amersham Life Science).

Immunofluorescence. Mouse kidney (CD-1 and C57BL/6J strains) was processed for immunofluorescence as described (34). C4 antibody fluorescence was optimal in tissue perfused with PBS and 4% paraformaldehyde. Tissue sections were treated with 0.2% Triton X-100 (C4 primary antibody) or 1% SDS (C9 primary antibody) in PBS for 5 min to expose antigenic sites (5). Tissue sections were incubated in 1% BSA/PBS/4% Seablock (Searun Holdings, Arundel, ME) for 30 min at room temperature. Slides were incubated overnight at 4°C with affinity purified anti-C4 antibody (1:100) or anti-C9 antibody (1:2,000) diluted in 1% BSA/PBS/4% SeaBlock. Sections were washed with PBS/2.8% NaCl followed by standard PBS, then incubated for 1 h with anti-rabbit Alexa 594 conjugate antibody (1:2,000) (Molecular Probes, Eugene, OR) diluted in 1% BSA/PBS/4% SeaBlock. Sections were washed as above and mounted with Vectashield mounting medium (Vector Labs, Burlingame, CA) diluted with equal parts 0.3 M Tris, pH 8.8. Slides were examined with a Nikon Eclipse 800 research microscope. Images were generated using an Optronics (OEI-750) video imaging system and printed using a Tektronix Phaser 450 dye-sublimation color printer.

Immunohistochemistry. Mouse kidney sections were obtained and treated as described above. Following blocking with 1% BSA/PBS/4% SeaBlock, the tissue was treated with avidin-biotin for 30 min at room temperature using a kit obtained from Vector Labs. The antibody was applied as before and incubated overnight at 4°C. The sections were allowed to come to room temperature before washing. Sections were washed with PBS/2.8% NaCl followed by standard PBS, then incubated for 1 h at room temperature with anti-rabbit biotinylated antibody (1:200). Sections were washed as above and incubated with Vectastain ABC peroxidase for 1 h followed by two 5-min PBS washes. The sections were then developed using Vector diaminobenzidine substrate kit and mounted with Aquapolymount (Polysciences, Warrington, PA). Slides were examined with a Nikon Eclipse 800 research microscope. Images were generated using an Optronics (OEI-750) video imaging system and printed using a Tektronix Phaser 450 dye-sublimation color printer.

Chromosomal localization and characterization of genomic clones. Primers from within the 3'-untranslated region (UTR) of the C9.0 cDNA were used to test for single-strand conformation polymorphisms (SSCPs) between mouse strains. These were analyzed as previously described (4, 7). The primer pair S6 5'-GGTATTAACCCATGCTTTACAG-3' and AS4 5'-GGCATATGTTGACTCTTTGGG-3', which amplifies nucleotides 4359 to 4546 of mBSC1-F9, identified a series of polymorphisms. Since these primers identified a polymorphism between the C57BL/6J and C3H/FeJ strains, they were used to analyze DNA prepared from the BXH recombinant inbred series.

To determine the mechanism of the alternative splicing that generated the C4 3' end, the corresponding segment of the mBSC1 gene was characterized. Three overlapping PCR fragments were generated from genomic DNA, spanning the exon encoding the C4 3' end and the two flanking introns (Fig. 4B). The three fragments were subcloned into the EcoR V site of pBluescript and sequenced; the entire C4 3' exon was sequenced on one strand, and sequences around exon/intron boundaries were determined on both strands.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Isolation and sequence of cDNA clones. Two major classes of mBSC1 clones, designated C4 (clone 4) and C9 (clone 9), were identified. Eight C4 clones and nine C9 clones were isolated. One full-length clone from each group, C4.0 (2,968 nucleotides) and C9.0 (4,605 nucleotides), was sequenced in entirety on both strands. The sequence of C4.0 has been deposited in GenBank (accession no. U61381) as mBSC1-A4, where "A" refers to the A exon cassette and "4" refers to the C4 3' end (see below); the C9.0 sequence was deposited as mBSC1-F9 (accession no. U94518).

The 5'-UTR of the two cDNAs are identical, although that of C4.0 extends a further 22 bases 5' of C9.0. The initiation ATG codon is located at nucleotide 202 in C4.0 and nucleotide 180 in C9.0, and the sequence of the C4.0 and C9.0 clones contain single open-reading frames of 2,310 nucleotides (770 amino acids) and 3,285 nucleotides (1,095 amino acids), respectively. The amino acid sequence of the C4.0 cDNA (mBSC1-A4) is displayed in Fig. 1A. The first 209 codons of the two clones encode identical amino acids, except for codon 11, which encodes a histidine in C9.0 and a proline in C4.0 (see below). After the cassette exons (see below), the 3' ends of the two clones are identical, until nucleotide 2345 in C4.0 and 2323 in C9.0. The last 165 nucleotides of the coding region of the C4.0 cDNA are distinct from that of the C9.0 cDNA, as is its predicted 3'-UTR. The 3'-UTRs of the C4.0 and C9.0 cDNAs are 445 and 1,130 bases long, respectively; both contain polyadenylation signals just 5' of the poly(A)+ tail.


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Fig. 1.   Sequences of full-length mBSC1 clones. A: deduced amino acid sequence of the mBSC1-A4 protein (C4.0 cDNA). COOH-terminal amino acids unique to this isoform are indicated with underscore (at bottom). Membrane spanning domains, predicted as in Ref. 10, are indicated by bold brackets (M1-M12), and the A cassette is enclosed by a box; black-diamond , predicted N-linked glycosylation sites; , predicted phosphorylation sites for protein kinase A (PKA); and open circle , protein kinase C (PKC) sites. B: deduced COOH-terminal amino acid sequence of the mBSC1 isoforms with the C9 3' end. Amino acids are depicted beyond the point where the mBSC1-A4 and mBSC1-F9 proteins diverge, for comparison to A. The last 14 amino acids common to both clones are indicated with underscore (at top). Predicted PKA and PKC sites are indicated as in A.

Three mutually exclusive exon cassettes, denoted A, B, and F, have been described near the 5' end of rabbit and mouse BSC1 partial cDNA clones (14, 24). The length of these exons in the mBSC1 gene is 96 nucleotides, encoding 31 amino acids (14). Eight different C4 clones (C4.0-4.7) were found to contain the murine A cassette, whereas the two longest C9 clones, C9.0 and C9.1, contained the F cassette. The A cassette extends from nucleotide 818-913 in C4.0, and the F cassette extends from nucleotides 796-891 in C9.0. The B cassette was independently cloned from mouse renal cortex by RT-PCR. The amino acid sequences of the three cassette exons are identical to those reported by Igarashi et al. (14).

The predicted membrane topology of the electroneutral cation-chloride cotransporter family (14, 21) is preserved in both of the full-length mBSC1 clones, with up to 12 hydrophobic potential membrane-spanning domains flanked by hydrophilic NH2- and COOH-terminal domains. The cassette exons are predicted to comprise most of the second transmembrane domain, extending 10-13 amino acids into a cytoplasmic loop. The C9 COOH-terminal domain, predicted to be cytoplasmic, is 457 amino acids in length; residues distinct from the C4 isoforms are displayed in Fig. 1B. In contrast, the cytoplasmic COOH-terminal domain of the C4 isoforms is 132 amino acids long, 325 amino acids shorter than that of the C9 isoforms, and the last 55 amino acids of C4 are distinct (Fig. 1A). The COOH-terminal 55 amino acids of the C4 isoforms have only limited homology to other proteins, including other members of the electroneutral cotransporter family.

The mBSC1-F9 isoform (C9.0 cDNA) shares 98% and 96% sequence identity with rBSC1-F and rabNKCC2-F, respectively. Although essentially identical to the previously reported mBSC1/NKCC2 sequence (14), it differs at a total of 49 nucleotides scattered throughout the sequence. Seven of these differences are nonconservative, predicting different amino acids (residues 11, 30, 48, 599, 773, 873, and 946). Three of these amino acids, residues 30, 48, and 599, are present and identical in the C4.0 sequence, and in all but one instance (the glycine at position 873) the predicted amino acid in the mBSC1 clones is identical to rBSC1 and rabNKCC2. However, partial mNKCC2 cDNA clones were derived from a different mouse strain (BALB/c vs. CD-1), and thus the discordance between the mBSC1 and mNKCC2 sequences may reflect polymorphism between strains. To investigate the variance between the C4.0 and C9.0 cDNAs at codon 11, nucleotides 73 through 238 of C9.0 were amplified by RT-PCR from CD-1 kidney (data not shown) and subcloned in the EcoR V site of pBluescript. Seventeen recombinant clones were picked at random and sequenced; eight cDNAs had CAC (histidine) at codon 11 and nine cDNAs had a CCC (proline) at this codon, suggesting polymorphism within the CD-1 strain.

The seven protein kinase C (PKC) consensus phosphorylation sites predicted from the rBSC1 sequence are conserved in C9, two in the NH2-terminal domain (Ser57 and Thr75) and five in the COOH-terminal domain (Thr639, Thr927, Ser983, Ser999, and Ser1029) (Fig. 1. A and B). Two of the three protein kinase A (PKA) sites in the COOH-terminal domain of rabNKCC2 and rBSC1 are preserved in the C9 COOH terminus (Ser1013 and Ser1062). Both PKA consensus sites and three out of the four PKC consensus sites present in the C9 COOH-terminal domain are absent in the C4 COOH terminus. There are two PKC sites (Ser756 and Thr761) and one PKA site (Thr761) in the C4 COOH-terminal 55 amino acids (Fig. 1A). The threonine residue at position 761 is a predicted phosphorylation site for both PKA and PKC.

Detection and characterization of mBSC1 transcripts. Northern blots of outer medulla RNA (inner stripe) were performed with splice form-specific probes and with a probe containing sequences common to both isoforms (see MATERIALS AND METHODS). The C4-specific probe detected transcripts of 3.0, 7.4, and >9.0 kb (Fig. 2A). The C9-specific probe detected a 4.6-kb transcript (Fig. 2B). A survey of several tissues (data not shown) with the probe common to all mBSC1 isoforms confirms that expression of mBSC1 is specific to the kidney, as reported previously (14).


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Fig. 2.   Northern analysis of mBSC1 transcripts. A: C4-related transcripts; 2.5 µg per lane of poly(A)+ RNA from murine kidney was probed at high stringency with a 382-bp C4-specific probe. Autoradiogram was exposed for 45 h. B: C9-related transcripts; 2.5 µg per lane of poly(A)+ RNA from murine kidney was probed at high stringency with a 475-bp C9-specific probe, and autoradiogram was exposed overnight.

RT-PCR was first used to demonstrate that the C4 and C9 clones are derived from transcripts expressed in mouse kidney, using antisense primers specific for each 3' end. Specificity was verified for each primer pair (shown for C4 in Fig. 3A). The C9 primer pair does not amplify genomic DNA, whereas the C4-specific pair reproducibly amplifies a 1.3-kb fragment from genomic DNA (Fig. 3A) because of the intervening intron of 750 bp (Fig. 4B). Both primer pairs amplify bands of the appropriate size from cortex and from inner and outer stripe of outer medulla (Fig. 3, B and C); identity was confirmed by subcloning and sequencing of PCR products.


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Fig. 3.   RT-PCR of kidney RNA. Ethidium-stained 6% polyacrylamide gels are displayed; one-fifth of each PCR reaction was loaded per lane. A: sensitivity and specificity of the C4-specific primer pair. Sense primer S1 and C4-specific antisense primer AS1 were tested on mBSC1 cDNA templates, mBSC2 cDNA, and on genomic DNA. B: RT-PCR of kidney with C4-specific primers; both RT-positive (RT+) samples and RT-negative (RT-) controls were amplified with C4-specific primer pair S1 and AS1 (amplification product 510 bp). C: RT-PCR of kidney with C9-specific primers S2 and AS2 (amplification product 278 bp). D: RT-PCR of kidney with cassette-specific sense primers and antisense primers specific for the C4 and C9 3' ends; +, RT+; -, RT-. Amplified cDNA was subcloned into pBluescript, generating the partial cDNA clones shown in Fig. 4A. mBSC1-B4 (1,602 bp) and mBSC1-B9 (1,692 bp) cDNAs were amplified from renal cortex. mBSC1-F4 cDNA (1,619 bp) was amplified from inner stripe (IS), and the mBSC1-A9 cDNA (1,668 bp) was amplified from outer stripe (OS) of outer medulla.

The combination of three 5' and two 3' alternative splicing events suggested a possible total of six different expressed isoforms, two of which were represented by the C4.0 (mBSC1-A4) and C9.0 (mBSC1-F9) cDNA clones. Previous reports do not address this issue, because the partial clones obtained did not link all of the 5' cassettes to 3' coding sequences (10, 14, 24). Partial cDNAs representative of the four remaining isoforms were cloned from poly(A)+ RNA by RT-PCR, using the appropriate combination of primers (Fig. 3D); water controls for each primer pair were negative, and the relevant cDNA templates yielded major PCR bands of the same size as RT+ RNA (data not shown). Higher molecular weight bands were also seen for mBSC1-F4 and mBSC1-A9; these uncharacterized bands are not due to genomic amplification but likely reflect amplification of other templates. Guided by the reported intrarenal distribution of cassette exon transcripts (24), the two isoforms containing the B cassette were amplified from superficial renal cortex. The mBSC1-F4 and mBSC1-A9 isoforms were cloned from inner and outer stripe of outer medulla, respectively. Bands of the appropriate size were subcloned into the EcoR V site of pBluescript, and 5' and 3' insert ends were sequenced to verify the specificity of each primer pair. Figure 4A summarizes the cDNA clones identified by library screening and RT-PCR.


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Fig. 4.   A: schematic representation of full-length and partial cDNA clones, showing relative positions of the cassette exons (letters), C4 3' end (hatched), and C9 3' end (shaded). The two full-length clones were obtained by library screening, and partial clones were obtained by RT-PCR. B: partial representation of the mBSC1 gene, encompassing alternatively spliced exons; exon 16 encoding the C4 3' end (hatched), exons encoding the C9 3' end (solid), and the cassette exons (indicated by letters B, A, and F). The order of the cassette exons, different forms of coding exon 4, is from Ref. 14. The section of the gene encompassed by the 3 overlapping genomic clones is expanded (not drawn to scale). Donor and acceptor sites are provided under each exon-intron boundary, with the nucleotides within exons in larger, bold font. The alternative polyadenylation site (AATAAA) in the intron between coding exons 16 and 17 is indicated by an arrow.

Chromosomal localization and genomic structure. SSCP analysis (4) was used to map mBSC1. Primers within the C9.0 3'-UTR were analyzed and found to identify an SSCP between inbred mouse strains. The BXH recombinant inbred series was genotyped and the strain distribution pattern analyzed using the Map Manager program (19). The mBSC1 gene was found to map to chromosome 2 with a LOD likelihood score of 3.6. No recombinants were found between mBSC1 and beta 2-microglobulin (6) in the 12 BXH strains (95% confidence interval: 0-18 cM). The mapping to mouse chromosome 2 is concordant with the localization determined using an informative Taq I restriction fragment length polymorphism (27).

Sequence comparison between the cDNA clones and genomic DNA (Fig. 4B) reveals an internal donor site, TGGTAAGAG (nucleotides 2342-2350 of C4.0), at the point at which the C4 and C9 clones diverge. This and other donor/acceptor sites in the genomic clones (Fig. 4B) conform to published consensus sequences (23). The exon encoding the C4 3' end is homologous to coding exon 16 of the human BSC1 gene, although it begins one codon later at the third nucleotide of codon 677. The alternative polyadenylation site found in the 3'-UTR of the C4.0 cDNA is identified within the intron between coding exons 16 and 17. Genomic PCR (data not shown) indicates that the C9 3' end is encoded by several exons, as demonstrated in the human gene (29).

Characterization of mBSC1 proteins. In vitro translation of the C4.0 and C9.0 cDNAs gave rise to proteins of molecular masses of 77 ± 5 and 131 ± 18 kDa, respectively, versus the predicted masses of 83 and 120 kDa (Fig. 5A). The broad bands obtained on SDS-PAGE of the in vitro translation products are also seen with rBSC1 (10). Like rBSC1, the molecular weight of mBSC1 proteins is not affected in vitro by the addition of pancreatic microsomal membranes (data not shown), despite the presence of potential N-linked glycosylation sites (Fig. 1, A and B).


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Fig. 5.   Characterization of mBSC1 proteins. A: in vitro translation of mBSC1 proteins. Autoradiography of a 10% SDS-PAGE gel showing the in vitro translation products of the C4.0 and C9.0 cDNAs, exposed overnight. B: Western blot of outer medulla membrane protein (125 µg, 6% SDS-PAGE) with the C9-specific antibody (ECL system, 30-s exposure). C: Western blot of membrane protein (150 µg/lane) from several tissues, including kidney (inner stripe of outer medulla), with immune serum generated against amino acids 735-749 of the predicted mBSC1 C4 isoforms (ECL system, 10-min exposure). D: Western blot of renal membrane protein (150 µg/lane for inner stripe and papilla, 250 µg/lane for cortex), using affinity-purified C4 antibody (1:150 titer, ECL, 2-min exposure) and immunoabsorbed ("immuno-ab.") affinity-purified C4 antibody (150 µg of inner stripe membrane protein, 1:150 antibody titer, ECL, 48 h exposure).

Western blots were performed with a polyclonal antibody that is predicted to react with C9 proteins and not C4 (see MATERIALS AND METHODS). This antibody reacts with a doublet of ~150 kDa, ~30 kDa heavier than the predicted core protein, in a membrane fraction of outer medulla (Fig. 5B). The use of a lower percentage polyacrylamide gel than in our previous study (16) permitted the resolution of the mBSC1 protein into two distinct bands, likely differentially glycosylated forms. Attempts to deglycosylate the rat and mouse proteins recognized by this antibody have not been successful (data not shown). However, both NH2-terminal (8) and COOH-terminal antibodies to BSC1 (16) recognize a protein of 150-160 kDa.

Western blotting was also performed with an antibody generated against a peptide from the unique COOH terminus of the C4 isoforms. One of two rabbits immunized with this antigen generated antiserum that reacts with a doublet of ~120 kDa (Fig. 5C). Again, attempted deglycosylation of this protein was not successful. All of the cation-chloride cotransporters are glycoproteins, and alternative splicing does not change the putative glycosylation sites in the extracellular loop of mBSC1 C4 isoforms. Because the molecular mass of the C9 isoforms is ~30-40 kDa higher than that of the core protein (Fig. 5B and Ref. 8), the expected molecular mass of glycosylated C4 proteins is in the range of 110-120 kDa, as seen with the C4 antibody. Affinity-purified antibody recognizes a protein of the same molecular weight in both cortex and outer medulla, and reactivity is abolished by preabsorption with antigen (Fig. 5D).

Intrarenal localization of mBSC1 COOH-terminal isoforms. Localization of mBSC1 COOH-terminal isoforms in mouse kidney was assessed using affinity-purified isoform-specific antibodies in immunohistochemistry, immunofluorescence (IF), and differential interference contrast with immunofluorescence (DIC-IF). The anti-C4 antibody detected mBSC1 C4 isoforms within TAL cells in the inner stripe of outer medulla (Fig.6A), and staining with antigen-absorbed antibody was negative (Fig. 6C). However, as shown by high-magnification DIC-IF images in Fig. 6, B and D, staining was heterogeneous with not all cells being labeled. In Fig. 6D, mBSC1 C4-positive and -negative cells within a tubule profile are indicated by white and black arrows, respectively. The qualitative degree of heterogeneity in outer and inner stripe of outer medulla appeared to be equivalent (data not shown). Reactivity to the anti-C4 antibody was also detected in the CTAL; however, the proportion of positive cells appeared to be lower than in outer medulla (Fig. 6E). As one progressed up individual tubule profiles into the cortex from the outer stripe, C4-positive cells became less frequent (Fig. 6E). Immunohistochemistry was more sensitive in detecting positive cells in CTAL (Fig. 6F); however, heterogeneous staining was still apparent.


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Fig. 6.   Immunolocalization of mBSC1 isoforms within mouse kidney. A: immunofluorescence (IF) of inner stripe of outer medulla (×400) stained with anti-C4 mBSC1 antibody. B: combined differential interference contrast (DIC) and IF of inner stripe stained with anti-C4 mBSC1 antibody (×600). Staining of tubular cells within thick ascending limb (TAL) is appreciated, with negative staining of outer medullary collecting duct (CD). C: negative DIC-IF (×400) of inner stripe of outer medulla stained with immunoabsorbed anti-C4 antibody. D: combined DIC-IF (×1,000) of inner stripe stained with anti-C4 antibody. In one tubular profile, black arrows indicate TAL cells with minimal staining and white arrows indicate strongly positive cells. E: combined DIC-IF (×1,000) of two medullary rays stained with anti-C4 antibody, extending from outer stripe of outer medulla (at bottom) into cortex. Positive cells are indicated by arrows. F: immunohistochemistry of a medullary ray stained with anti-C4 antibody. Positive cells are indicated by arrows. G: IF (×400) of inner stripe stained with anti-C9 mBSC1 antibody. H: IF (×400) of cortex stained with anti-C9 mBSC1 antibody. I: combined DIC-IF (×600) of inner stripe stained with anti-C9 antibody.

In comparison with the results with anti-C4 antibody, labeling of mouse TAL with the anti-C9 antibody was much more uniform, in both outer medulla and cortex (Figs. 6, G and H), as previously shown in the rat (16, 34). Labeling of the apical membrane was also very distinct (Fig. 6I). In contrast, although labeling with the anti-C4 antibody showed apical predominance, subapical labeling was also seen (Fig. 6, A, B, D, and E).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We report here the isolation and sequence of two full-length cDNAs, designated mBSC1-A4 (C4.0, 3.0 kb) and mBSC1-F9 (C9.0, 4.6 kb), encoding the mouse apical or absorptive Na+-K+-2Cl- cotransporter mBSC1. The two clones are generated by alternative splicing and differ in the incorporation of both 5' and 3' exons. The predicted membrane topology of both proteins is identical to other members of this family, and mBSC1-F9 is the murine homolog of rBSC1 and rabNKCC2. The shorter cDNA, mBSC1-A4 (C4.0 clone), predicts truncation of the COOH-terminal cytoplasmic domain by 325 amino acids, with substitution of 55 new amino acids at the extreme COOH-terminal end.

Using both Northern blot analysis and RT-PCR, we have demonstrated that transcripts corresponding to the identified cDNA clones are expressed within mouse kidney. Northern analysis of outer medulla mRNA with the C9-specific probe reveals the expected 4.6-kb transcript. In addition to a 3.0-kb transcript corresponding to the C4.0 cDNA, two transcripts of 7.4 kb and >9 kb were detected in inner stripe of outer medulla using a C4-specific probe. The longer transcripts presumably differ in the length of the 3'-UTR. Although formal quantitation was not performed, C4-related transcripts are apparently expressed at much lower levels than C9 transcripts.

SSCP analysis was used to localize mBSC1 to mouse chromosome 2, tightly linked to beta 2-microglobulin; this mapping is consistent with that reported by Quaggin et al. (27), which it confirms by an independent method. Characterization of a 3' portion of the mBSC1 gene, encompassing the murine equivalent of human coding exon 16 (29), reveals an internal donor site in the coding sequence of the 3' end of the C4 isoforms (Fig. 2B). The bypass of this donor site and utilization of an alternative polyadenylation site, known mechanisms of alternative splicing (30), generate the unique 3' end of the C4 isoforms. The three cassette exons can pair with both alternative 3' ends, such that a total of six different isoforms were identified (Figs. 3D and 4A). The two splicing events are thus presumably independently regulated. The expression of the A, B, and F cassettes appears to be spatially restricted along the TAL (in outer stripe, cortex, and inner stripe, respectively) (14, 24). The C4 protein is detected at the apical membrane of the TAL (see below), thus the combined data indicate that cells in all segments of mouse TAL have the potential to express both COOH termini paired with segment-specific exon cassettes.

To verify that the C4 isoforms are in fact translated in vivo, an important test for the relevance of alternatively spliced mRNAs, a peptide-specific C4 antibody was generated in rabbits. Western blotting with affinity-purified C4 antibody detects a protein of 120 kDa in a membrane fraction from outer medulla, and reactivity is abolished by preabsorption with antigen. This molecular weight corresponds to glycosylation of an 80-kDa core protein, because glycosylation of other members of the cation-chloride cotransporter family adds ~30-40 kDa to the molecular mass of the transporter proteins (15, 18, 26). A previous study with an NH2-terminal BSC1 antibody, predicted to react with both C9 and C4 isoforms, did not report a protein of the molecular weight identified by the C4 antibody (8). Potential reasons for this discrepancy include the relative amount of membrane protein per lane [1-3 µg in Ecelbarger et al. (8) vs. 150 µg in this study].

Western blotting of outer medulla with the C9 antibody detects a protein of ~150 kDa, the same size as the high-affinity bumetanide-binding protein detected in mouse outer medulla. The C9 protein is expressed at the apical membrane of epithelial cells in both the CTAL and MTAL. Expression in Xenopus oocytes of the three exon cassettes in the context of the longer C9 3' end results in bumetanide-sensitive, interdependent uptake of Na+-K+-Cl- (see companion study). The observation that all three are active in K+ transport indicates that the cassette exons do not contribute directly to K+-independent Na+-Cl- cotransport (31).

The C4 antibody also labels the apical membrane of TAL cells. However, labeling is much more heterogeneous in all segments of the TAL, such that not all cells are positive. This is particularly true in CTAL (Fig. 6, E and F), and staining tends to decrease in frequency as one moves up individual medullary rays toward the cortex. The heterogeneity of staining within inner and outer stripe was roughly equivalent. Phenotypic heterogeneity of TAL is also detected with antibodies to the K+ channel ROMK, which do not react with a subpopulation of rat TAL cells (34). Electron microscopy of rat TAL has previously identified two subtypes of TAL cells, "rough" and "smooth," which differ in the relative abundance of apical microvilli (1). In the hamster TAL, the relative frequency of these two cell types correlates closely with the proportion of cells having high and low apical K+ conductance (32). The issue of which morphological subtype of TAL cells expresses ROMK and the mBSC1 C4 isoforms will require further study at the electron microscopy level.

In addition to the observed cellular heterogeneity, staining of TAL with the C4 antibody is not as sharply apically defined as that produced by the C9 antibody, suggesting a significant component of subapical expression (Fig. 6, A, B, D, and E). This will need to be confirmed by immunoelectron microscopy. BSC1 protein is detectable in a population of subapical vesicles (22), and trafficking of BSC1-containing vesicles to the apical membrane may be involved in regulation of salt transport in the TAL. The amino acids removed and/or added to the COOH termini of the C4 isoforms may affect the interaction of mBSC1 with components of subapical vesicles and/or the cytoskeleton (9). Coexpression of C9 and C4 isoforms in Xenopus oocytes reveals a dominant negative role for the truncated C4 isoforms, an effect that is reversed by cAMP (25). The dominant negative effect of the C4 isoforms may depend on differential sorting to the plasma membrane. Specifically, since BSC1 proteins evidently form multimers (17), membrane trafficking may be attenuated for C9 isoforms "trapped" by coassociation with C4 isoforms. Alternatively, the C4 COOH terminus may function to block ion permeation through the transporter complex, analogous to the effect of the COOH terminus of the ENaC beta -subunit on the open probability of epithelial Na+ channels (3).

Alternative splicing at the 3' end of mBSC1 alters the consensus sites for PKC- and PKA-dependent phosphorylation (Fig. 1, A and B). The C4 protein contains a cluster of potential phosphorylation sites in the last 20 amino acids of its unique COOH terminus, with two PKC sites and one PKA site not found in C9. In addition, three of four PKC sites and the two PKA sites in the COOH terminus of C9 are absent in C4. Similar modulation of predicted phosphorylation sites occurs in mBSC2, for which alternative splicing of exon 21 creates isoforms that lack a PKA site (28). Although there is as yet no data on the direct phosphorylation of mBSC1, several hormones activate adenylate cyclase and PKA in mouse TAL, with subsequent stimulation of ion transport. Full-length C9 isoforms are not activated by cAMP when expressed in Xenopus oocytes (25). However, coexpression with C4 isoforms reconstitutes a response of mBSC1 isoforms to cAMP. The observed decrease in the frequency of C4-positive cells in the mouse CTAL may explain the relative resistance of this segment to vasopressin (13) because the lower abundance of mBSC1 isoforms with a C4 COOH terminus would affect stimulation of ion transport by cAMP.

In summary, a truncated mBSC1 COOH terminus is generated by alternative splicing of the intron between exons 16 and 17. The combinatorial association of both truncated (C4) and full-length (C9) 3' ends with three alternative versions of coding exon 4 (cassette exons A, B, and F) generates a total of six alternatively spliced isoforms. Both truncated and full-length isoforms are translated in mouse kidney and are coexpressed within TAL. As demonstrated in the companion study (25), truncated C4 isoforms are not functionally active in Xenopus oocytes. However, these isoforms have a dominant negative effect when coexpressed with the longer isoforms. This effect is reversed by cAMP, suggesting a role for such interactions in regulating salt transport within TAL.


    ACKNOWLEDGEMENTS

This work was supported by grants from the National Institutes of Health to S. C. Hebert (DK-36803), D. R. Beier (HD-29028 and HG-00951), and D. B. Mount (DK-02103) and by grants from the Mexican Council of Science and Technology (CONACYT, M3840) and Howard Hughes Medical Institute (75197-553601) to G. Gamba. A. Baekgaard was supported by the Danish Research Council (11-9735, 11-0272, 11-0897) and by the King Kristian the Tenth Foundation (0204-0096-006317). C. Plata was supported by scholarship grants from CONACYT and DGAPA.


    FOOTNOTES

Portions of this work were presented at the Annual Meetings of the American Society of Nephrology and have been published in abstract form (J. Am. Soc. Nephrol. 5: 282, 1994; J. Am. Soc. Nephrol. 6: 347, 1995; and J. Am. Soc. Nephrol. 7: 1288, 1996).

D. B. Mount and A. Baekgaard contributed equally to this study.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: D. B. Mount, Renal Division, Vanderbilt Univ., MCN S-3223, Nashville, TN 37232 (E-mail: david.mount{at}mcmail.vanderbilt.edu).

Received 15 June 1998; accepted in final form 30 October 1998.


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DISCUSSION
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