Functional and molecular characterization of the shark renal Na-K-Cl cotransporter: novel aspects

Édith Gagnon1, Biff Forbush2,3, Andreas W. Flemmer2, Ignacio Giménez2, Luc Caron1, and Paul Isenring1

1 Groupe de Recherche en Néphrologie, Department of Medicine, Faculty of Medicine, Laval University, Laval, Quebec, Canada G1R 2J6; 2 Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510; and 3 Mount Desert Island Biological Laboratory, Salisbury Cove, Maine 04672


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The Na-K-Cl cotransporter isoform 1 (NKCC1) has been isolated from several species, including Squalus acanthias. A second kidney-specific isoform (NKCC2) has been cloned mainly from higher vertebrates. Here, we have isolated the S. acanthias NKCC2 and found that it is produced in at least four spliced variants (saNKCC2A, saNKCC2F, saNKCC2AF, and saNKCC2AFno8) of ~1,090 residues. Expression of these transcripts in Xenopus laevis oocytes revealed that only the A and F variants are functional and that they are more active after incubation in low-Cl or hyperosmolar media. Rates of activation after exposure to these media were exceptionally rapid, demonstrating for the first time that the NKCC2 itself represents an important site of regulation by Cl and that extracellular domains are involved. Another remarkable finding in this study was the failure to identify NKCC2B, a variant found in the kidney of higher vertebrates and expressed specifically in macula densa cells. This result, in conjunction with the fact that the shark kidney lacks a well-developed juxtaglomerular apparatus, suggests that the B exon evolved as a result of selective pressure (presumably by exon duplication) and that a restricted relationship exists between NKCC2B and macula densa.

cation-chloride cotransporter; squalus acanthias; splice variant; thick ascending loop of Henle


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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TWO ISOFORMS of the Na-K-Cl cotransporter (NKCC) have been identified: NKCC1, which has wide tissue distribution in vertebrates (15, 16, 34, 51), and NKCC2, which is kidney specific (17, 20, 39). The NKCCs belong to the cation-Cl cotransporter (CCC) family, which also includes the K-Cl cotransporters (KCCs) (10, 36, 40, 45), the Na-Cl cotransporter (9), and a CCC-interacting protein (CIP1) (4, 23). A phylogenetic representation of this family is shown in Fig. 1.


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Fig. 1.   Cladogram of cation-Cl cotransporter (CCC) proteins. A: phylogenetic tree, which includes Na-K-Cl cotransporter isoforms 1 and 2 (NKCC1 and NKCC2) from 4 different species, constructed using MEGALIGN (Lasergene). To include the rabbit (rb) NKCC1 sequence, the cladogram is based on a 175-residue region between the end of the NH2 terminus and the end of transmembrane (tm) region 6 (tm6 region). B: phylogenetic tree constructed with entire coding region of 10 different CCCs. hu, Human; ms, mouse; sa, Squalus acanthias; x, unknown ionic substrate. * Also called huCIP1 (4, 23).

The NKCC plays an important role in maintaining the water and electrolyte content of individual cells (15, 24) and in promoting salt and water movement across polarized cells (13, 24, 50). The cotransport process is activated by phosphorylation of the transporter after a decrease in extracellular Cl or an increase in extracellular tonicity. It has been hypothesized that these stimuli activate the NKCCs through a decrease in intracellular Cl or a decrease in cell volume, respectively (16, 33).

In mammals, three different 96-bp exons encoding transmembrane (tm) region 2 (tm2 region) of NKCC2 are alternatively spliced, generating three variants: A, B, and F (9, 39). In situ hybridization (20, 39) and immunolocalization studies (29, 52) have revealed that these variants are confined to the thick ascending limb of Henle (TAL), where they are distributed as follows: A in cortex and medulla; B in macula densa cells, playing a potential role in tubuloglomerular feedback (TGF); and F in medulla. A fourth variant, which has been described in the rat and rabbit kidney, consists of a medullary NKCC2 containing the A and F exons in tandem (17, 39). The role of the alternatively spliced 96-bp region has been recently investigated (6, 7, 11, 12, 43).

The evolutionary dating of NKCC2 relative to the phylogenetic development of the kidney has not been determined. Biemesderfer et al. (2) recently demonstrated that NKCC is present in the lateral bundle zone of the elasmobranch kidney, supporting the concept that these structures are functional equivalents of mammalian TALs and are able to induce countercurrent exchange. However, the distal segment of the elasmobranch TAL appears poorly developed, lacking morphologically distinct macula densa cells (38) and the capacity to induce changes in glomerular filtration through the TGF mechanism (31).

In this study, we have identified three known NKCC2 variants in the Squalus acanthias kidney: saNKCC2A, saNKCC2F, and saNKCC2AF. We have also identified a new variant called saNKCC2AFno8. Functional analyses of these proteins expressed in Xenopus laevis oocytes demonstrate that saNKCC2AF and saNKCC2AFno8 are inactive. The saNKCC2A and saNKCC2F, on the other hand, induce 86Rb fluxes after preincubation in low-Cl or hypertonic medium. We have also found that the S. acanthias gene does not contain the B exon.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Cloning of saNKCC2. A 520-bp fragment encoding the tm5-tm7 region of the NKCCs was amplified by RT-PCR using poly(A) RNA isolated from S. acanthias kidneys and degenerate primers (oligos 1 and 2; Table 1) derived from conserved NKCC regions (bp 1632-1654 and 2127-2149 in saNKCC1). This fragment was then random labeled with 32P to screen a custom-made lambda ZAPII S. acanthias kidney cDNA library. In the first round of screening, signals were noted for ~7 × 102 of ~5 × 106 plated plaques. Seventy-two positive regions were isolated (primary stocks), and clonal phages were obtained from nine of these stocks through additional rounds of screening. Restriction analyses of phage-excised inserts revealed that all cDNAs, denoted "we are in business again" (WIBA) x.x, were unique.

                              
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Table 1.   Oligonucleotides

The longest clone among these 9 WIBAs (WIBA 7.1) was sequenced bidirectionally. It was found to contain a 192-bp 5'-untranslated region (UTR), a 3,273-bp open reading frame, and a 899-bp 3'-UTR (Fig. 2). The deduced amino acid sequence (1,091 residues) corresponds to the lagomorph NKCC2AF (17, 39), except a 108-nucleotide fragment is missing; this fragment, which would extend from bp 1723 to 1830, was arbitrarily termed cassette 8, because it encodes the tm8 region and parts of the tm7-tm8 connecting loop; accordingly, WIBA 7.1 is now termed saNKCC2AFno8.


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Fig. 2.   Cloning of saNKCC2. Alignment of 8 "we are in business again" (WIBA) clones isolated from S. acanthias cDNA library after several rounds of screening is shown. Clones that end with an open box were not sequenced beyond that region.

For six of the nine WIBAs (14.1, 11.2, 9.2, 9.1, 4.2, and 3.2), partial sequencing of the tm2 region revealed that two were AF variants and four were F variants. For WIBAs 14.1, 9.2, and 9.1, in addition, sequencing of the tm8 region also showed that two had a cassette 8 and one ended before bp 1723 (on the basis of WIBA 7.1). These WIBAs are shown in Fig. 2, tentatively aligned to WIBA 7.1; because only one cDNA was fully sequenced, the possibility of additional differences among clones cannot be ruled out. For the last two WIBAs (17.2 and 13.1), restriction analyses showed that one corresponded to NKCC1 and one had artifacts.

To identify putative A and B variants, as well as additional no8 variants, we used gene-specific primers (Table 1) and amplified the tm2 (oligos 3 and 4) and/or the tm8 (oligos 5 and 6) region of saNKCC2s from several primary stocks. Through size and restriction analyses, it was possible to categorize the PCR products as being from F (AflII site, 482 bp), AF (AccI and AflIII sites, 578 bp), A (AccI site, 482 bp), B (no AccI or AflIII site,1 482 bp), no8 (398 bp), or +8 (506 bp) variants. On the basis of this categorization, a 10th primary phage stock was selected to obtain an A variant; the resulting clone (~4 kb) is also called WIBA 7.8 (Fig. 2).

Southern analyses. To determine whether deletion of cassette 8 was an artifact, this region was amplified from the library using oligos 5 and 6, which hybridize to regions near cassette 8 (<140 bp on each side); here, the saNKCC2AF+8 and saNKCC2AFno8 cDNAs were used as controls. DNA fragments were probed as previously described (41), with a [gamma -32P]ATP-labeled primer called oligo 7 (Table 1); this probe hybridizes to a region immediately following oligo 5.

Amplification of genomic DNA. To determine whether a B exon is present in the S. acanthias NKCC2 gene, we amplified an appropriate region using ~1 µg of genomic DNA (kindly provided by Dr. Thomas Singer, Waterloo Biotelemetry Institute, Dept. of Biology, University of Waterloo, Waterloo, ON, Canada) and a pair of cDNA-based primers, oligos 8 and 9 (Table 1), derived from conserved NKCC regions. Here, the 5' primer (oligo 8) hybridizes to exon 3 and the 3' primer (oligo 9) to the middle of exon A. The amplified fragment (saG3.4) was sequenced directly.

cDNA constructions. The full-length saNKCC2AFno8 was used as template to generate six constructs. The PflmI-BspEI fragment of saNKCC2AFno8 (~1.6 kb) was replaced by that of WIBA 3.2 (~1.7 kb) to generate saNKCC2AF+8. Then, the SphI-PflmI fragment of saNKCC2AF (~0.8 kb) was replaced by that of WIBA 7.8 or 9.2 (~0.7 kb) to generate saNKCC2A or saNKCC2F. Two other constructs, saNKCC2Ano8 and saNKCC2Fno8, were obtained by replacing the SphI-PflmI fragment of saNKCC2AFno8 with that of WIBA 7.8 or 9.2; it is not known whether these two variants occur naturally.

For functional expression in HEK-293 cells, saNKCC2 cDNAs were transferred as KpnI-SmaI inserts into pJB20M (25) between a KpnI site and a blunted AvrII site; pJB20M is an expression vector derived from pCB6 (1). For functional studies in oocytes, the cDNAs were transferred as EcoRI inserts into Pol1, a vector that contains the X. laevis beta -globin UTRs and a poly(A) tract (4). For some of the studies, the rabbit (rb) NKCC2A (39) was also subcloned into Pol1 between EcoRI sites, and the SphI-BglII fragment (~0.7 kb long), which encodes tm2, was left in place or replaced by that of rbNKCC2F.

Expression of NKCC2s in HEK-293 cells and X. laevis oocytes. HEK-293 cells were transfected with 40 µg of cDNA by calcium phosphate precipitation and selected for geneticin resistance (950 µg/ml), as previously described (21, 25-27). Cell lines were maintained in DMEM (GIBCO), continuously supplemented with geneticin (950 µg/ml). For expression in oocytes, the cDNA constructs were in vitro transcribed with T7 RNA polymerase using the mMESSAGE mMACHINE T7 kit (Ambion). Defolliculated stage V-VI oocytes were injected with 25 nl of H2O or ~5-25 ng of cRNA diluted in 25 nl of H2O and assayed 3-5 days after injection.

Immunofluorescence studies. Immunolocalization of saNKCC2 in oocytes was performed as previously described (4). Briefly, egg cryosections (10 µm) were postfixed for 10 min in acetone, incubated overnight at 4°C with the anti-P-NKCC antibody, and detected with rhodamine-conjugated sheep anti-rabbit anti-IgG. Anti-P-NKCC is specific to the phosphorylated form of NKCC1 and NKCC2 (unpublished data) (5).

Functional studies. HEK-293 cells were transferred to 96-well plates coated with poly-D-lysine and grown to confluence at 37°C in DMEM (with G418). Oocytes were maintained for 3-4 days at 18°C in Barth's medium with 125 µM furosemide.

Ion transport rates were determined by 86Rb influx measurements at ~22°C using different media (Table 2). HEK-293 cells or oocytes were incubated first in tracer-free media during variable time intervals. Then the cells were incubated for up to 2 min (HEK-293) or 45 min (oocytes) in different media containing 86Rb (1-2 µCi/ml) and 10 µM ouabain with or without 250 µM bumetanide. Fluxes were terminated with washes in 250 µM bumetanide and 10 µM ouabain. For some experiments, osmolality of the media was adjusted with sucrose or urea; Na and K were replaced by N-methyl glucamine and Cl by SO4 and/or gluconate.

                              
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Table 2.   Composition of flux solutions

After the final washes, cells were solubilized in 2% SDS and 86Rb was detected by liquid beta -scintillation counting using the TopCountNXT microplate counter (Packard). Flux rates are presented as means ± SE. When appropriate, differences between groups of variables were analyzed by Student's two-tailed t-tests, and the null hypothesis was rejected for P < 0.05.

DNA sequencing and analysis. DNA fragments were subjected to automated sequencing. DNA analyses and tree constructions were carried out with WINSTAR and GCG programs.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cloning of saNKCC2. The isolation of several saNKCC2 clones from an S. acanthias kidney cDNA library (Fig. 2) led to the identification of four splice variants. Two such variants, saNKCC2A and saNKCC2F, differ from one another by a 96-bp region encoding the tm2 region. Another variant, saNKCC2AF, has the A and F cassette exons in tandem and is similar to that described in the lagomorph (17, 39). The fourth variant, saNKCC2AFno8 (or WIBA 7.1), is also an AF tandem form, except it lacks a 108-nucleotide segment encoding the tm8 region.

Multiple sequence alignments using NKCCs from several species indicate that saNKCC2A shares 59% identity with saNKCC1 and ~68% identity with the human (hu) NKCC2A or the mouse NKCC2A. The phylogenetic relationships between NKCC1s and NKCC2s and between NKCCs and other members of the family (KCCs, NCC, KCC, and CIP1) are illustrated in Fig. 1. Interestingly, the NKCC2s are as conserved as the NKCC1s (on the basis of a 175-residue region between the end of the NH2 terminus and the tm6 region).

Topology analyses of saNKCC2F predict a 12-transmembrane region flanked by cytoplasmic termini. Two candidate sites for N-linked glycosylation (residues 441 and 451) are found in a ~55-residue loop following the tm7 region. Consensus sites for protein kinase A (PKA), protein kinase C (PKC), tyrosine kinase (TK), and casein kinase II (CK) phosphorylation are present in the NH2 terminus (1 PKC, 1 TK, and 2 CK) and the COOH terminus (1 PKA, 6 PKC, 2 TK, and 5 CK); 10 of these 18 sites are conserved. For saNKCC2AF, saNKCC2AFno8, and saNKCC2Ano8, topology analyses predict structures that are very different, e.g., 11 transmembrane regions for saNKCC2Ano8, 13 transmembrane regions for saNKCC2AF+8, and an extracellular COOH terminus for both variants.

Alternative splicing of NKCC2 in the tm2 region. As indicated in MATERIALS AND METHODS, the tm2 region of several saNKCC2 clones (41 total) was characterized by sequencing or restriction analyses. Six of 41 clones (15%) corresponded to A variants, 24 of 41 (58%) to F variants, and 11 of 41 (27%) to AF variants. It was not possible to detect any B variant as found in the mouse, rabbit, rat, or human kidney.

To determine whether an saNKCC2B transcript was expressed at low levels in the kidney or whether there was no B exon in the S. acanthias DNA, we amplified a segment of the gene between exon 3 (oligo 8) and exon A (oligo 9). In the rodent gene, the corresponding segment includes (5' to 3') part of exon 3, a ~4.5-kb intron, exon 4B, a ~500-bp intron, and part of exon 4A (6, 38). We were able to amplify a 3.4-kb fragment (saG3.4) and determine its sequence. Interestingly, the region between exon 3 and A was devoid of any additional exons and aligned poorly (<55% identity) with that of the mouse gene (results not shown).

To determine whether the A, B, and F exons are phylogenetically related and whether the A or the F exons are conserved, we carried out multiple alignment studies using shark, mouse, rabbit, and human sequences. Results indicate that exon A shares, on average, ~64% identity with exon F (Fig. 3A) and that exon B is closer to exon A than it is to exon F (Fig. 3B). Interestingly, exons A and F are highly conserved from shark to human (~86% identity), especially in the COOH-terminal half (~93% identity), which corresponds to the intracellular loop following the tm2 region; this loop is the region of greatest conservation in the CCC family.


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Fig. 3.   Analysis of exon encoding the tm2 region. A: percent homology between the same exons and between A and F exons in shark, mouse, rabbit, and human. B: phylogenetic analysis of the tm2 region depicting distances between alternatively spliced exons in shark and rabbit. Tree includes 2 exons from shark and 3 from rabbit.

Alternative splicing in the tm8 region. As for the tm2 region, the tm8 region of several saNKCC2 clones (28 total) was characterized by sequencing or restriction analyses. We found that 3 of 28 clones ended before cassette 8, 23 of 28 had a cassette 8, and 2 of 28 (7%) were saNKCC2no8 variants. PCR studies using primers flanking the tm8 region showed that such variants constituted ~5% of the clones (Fig. 4). Here, fragments were generated from the library (lane 1) or from plasmids used as positive controls (lanes 2 and 3).


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Fig. 4.   Amplification of the tm8 region using oligos 5 and 6. A: gel electrophoresis of amplified fragment; in lane 1, S. acanthias cDNA library was used as template; in lanes 2 and 3, templates were saNKCC2AFno8 and saNKCC2AF+8 cDNAs, respectively. Approximately 1,000 ng of library-derived PCR products and ~50 ng of plasmid-derived products (amplified from ~1 pg of cDNA) were used for Southern blot. Expected sizes of amplified fragments are 398 bp (saNKCC2AFno8) and 506 bp (saNKCC2AF+8). B: Southern analysis, with oligo 7 used as an internal probe to confirm identities of bands in A.

Functional expression of saNKCC2s in HEK-293 cells. 86Rb transport rates (up to 2-min fluxes in basic medium) were measured in HEK-293 cells transfected with saNKCC2A, saNKCC2F, saNKCC2AF, saNKCC2Ano8, saNKCC2Fno8, or saNKCC2AFno8 after preincubation in different media (Table 2). It was not possible to detect above-background 86Rb transport for any of these variants (data not shown). These results are consistent with those reported for various mammalian NKCC2s (wild-type rbNKCC2A,2 rbNKCC2F, and rat NKCC2) expressed in mammalian cell lines (17, 39).

Functional expression of saNKCC2A and saNKCC2F variants in X. laevis oocytes. When saNKCC2A- or saNKCC2F-injected eggs were preincubated in an isosmolar normal-Cl solution and fluxed for 45 min in an isosmolar solution (Fig. 5A, bars 1, 5, and 9), 86Rb influx increased severalfold above background (14-fold for the F variant and 9-fold for the A variant). In these studies, the transport activities were >90% bumetanide sensitive (results not shown).


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Fig. 5.   Effect of hyperosmolality on 86Rb influx by Xenopus laevis oocytes injected with H2O, saNKCC2A, or saNKCC2F. A: oocytes were incubated for 45 min in basic medium (bm), basic medium + 84 mosM sucrose, basic medium + 84 mosM urea, or basic medium + 10 µM forskolin. Oocytes were subsequently assayed for 86Rb influx in basic medium + 10 µM ouabain. Values are means ± SE of 6-7 oocytes from 2 representative experiments. * P = 0.03; ** P < 0.002. B: oocytes were incubated for 45 min in a hyperosmotic medium and subsequently assayed for 86Rb influx in basic medium + 10 µM ouabain. Values are means ± SE of 4-6 oocytes from 1-5 experiments. Bu, bumetanide. C: 86Rb fluxes for saNKCC2F-injected oocytes were measured in basic medium after preincubation for 5-180 min in hyperosmolar medium (284 mosM) and were compared with those for H2O-injected oocytes. Values are means ± SE of 4-11 oocytes from 2 representative experiments. See Table 2 for composition of media.

Augmenting osmolality of the preincubation medium from 200 to 284 mosM with sucrose, an osmolyte that does not diffuse readily across the oocyte surface, led to a further increase in saNKCC2A and saNKCC2F activity (Fig. 5A, bars 6 and 10). In these experiments, 86Rb activity was also over ~90% bumetanide sensitive and clearly above that of endogenous CCCs (Fig. 5B). Replacing sucrose by urea, another osmolyte that does not diffuse readily across the oocyte membrane (49), resulted in a similar activation of saNKCC2s (Fig. 5A, bars 7 and 11). These results indicate that the effect of sucrose was due to a change in extracellular osmolality.

The mechanism by which an increase in extracellular osmolality stimulates NKCC was next examined by determining the time course of activation in hyperosmolar medium and the effect of forskolin in isosmolar medium. Data for saNKCC2F in Fig. 5C show that the time course is relatively short [deduced half-life (t1/2) < 30 min], paralleling that of a change in oocyte volume after an increase in extracellular osmolality (28). This result suggests that a hyperosmolar medium stimulates NKCC through cell shrinkage. No increase in 86Rb flux could be observed in forskolin-treated oocytes (Fig. 5A, bar 8 vs. bar 5 and bar 12 vs. bar 9). Hence, the effect of cell shrinkage on NKCCs does not appear to involve PKA-dependent mechanisms, in contrast to previous suggestions (14, 42).

Lowering extracellular Cl from 86 to 5.3 mM during preincubation also led to increased 86Rb transport by saNKCC2A- and saNKCC2F-injected eggs (2.7- and 4.7-fold, respectively; Fig. 6, A and B, bar 1 vs. bar 2 and bar 5 vs. bar 6). In Fig. 6, A and B, fluxes are higher under basal conditions in saNKCC2A (bar 1) than in saNKCC2F (bar 5), accounting for the more pronounced effect of low Cl on saNKCC2F (Fig. 5A, bar 5 vs. bar 9). Interestingly, a change in extracellular Cl had a less pronounced effect than a change in extracellular osmolality for saNKCC2F, whereas the effect of either change was similar for saNKCC2A. In Fig. 6, A and B, a decrease in extracellular Cl had no additional effect at higher osmolalities.


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Fig. 6.   Influence of external Cl concentration and osmolality on 86Rb influx by X. laevis oocytes injected with NKCC2 splice variants. In A, B, D, and E, oocytes were incubated for 45 min in basic medium (bars 1, 5, 9, and 13), a low-Cl isosmotic medium (bars 2, 6, 10, and 14), a hyperosmotic medium (bars 3, 7, 11, and 15), or a low-Cl hyperosmotic medium (bars 4, 8, 12, and 16). Oocytes were subsequently assayed for 86Rb influx in basic medium + 10 µM ouabain. Values are means ± SE of 5-12 oocytes from 3-6 experiments. Counts are normalized to data obtained at 284 mosM and to 86Rb concentration in flux medium. * P = 0.03; ** P < 0.002; ns, not significant. C: 86Rb fluxes for saNKCC2F-injected oocytes were measured in basic medium after preincubation for 5-180 min in isosmolar low-Cl medium (200 mosM, 5.3 mM Cl) and compared with those for H2O-injected oocytes. Values are means ± SE of 11-12 oocytes. See Table 2 for composition of media.

The mechanism by which a decrease in extracellular Cl stimulates NKCC is unknown. In HEK cells, we previously reported that low extracellular Cl led to full activation of huNKCC1 and hu1rb2A0.7 (the huNKCC1-rbNKCC2A chimera) within 45 min (t1/2 ~12 and ~2 min, respectively) (25). We have also measured time courses for saNKCC2-injected oocytes, and results are shown in Fig. 6C for the F variant. Interestingly, deduced t1/2 values were relatively similar to those reported above (~30 min), suggesting that activation in low extracellular Cl does not occur through changes in intracellular Cl; indeed, the low surface-to-volume ratio of the oocyte precludes rapid equilibration of intracellular with extracellular Cl (46). It is also seen that the time course shown in Fig. 6C exhibits two phases; the significance of this behavior, which was not observed for saNKCC2A (results not shown), is unknown.

Functional expression of rbNKCC2A variants in X. laevis oocytes. To determine whether the activating maneuvers described above exerted the same effect on NKCC2s from other species, rbNKCC2A- or rbNKCC2F-injected oocytes were also preincubated in low-Cl or hyperosmolar media (Fig. 6, D and E). Lowering Cl had no additional effect (bars 9 and 10 for rbNKCC2A, bars 13 and 14 for rbNKCC2F), the carriers being already activated in 86 mM Cl. Similarly, transport activities were not significantly increased by hyperosmolar preincubation (bars 11 and 12 for rbNKCC2A, bars 15 and 16 for rbNKCC2F).

Functional expression of other NKCC2 variants. As discussed above, our cloning efforts led to the discovery of a new variant in which a coding region is lacking. We also found that the AF variant was very abundant in the S. acanthias kidney, corresponding to ~30% of the NKCC2 transcripts. However, when these variants (Ano8, Fno8, AFno8, and AF+8) or when the rbNKCC2AF were injected in oocytes, it was not possible to detect 86Rb activity above that of H2O-injected controls (results not shown) regardless of the preincubating conditions.

Immunofluorescence studies in the oocyte showed that several of these variants were not expressed at the cell surface (Fig. 7D). Surprisingly, however, the signal distribution of saNKCC2AF+8-injected eggs (Fig. 7C) was similar to that of saNKCC2A-injected eggs (Fig. 7B). Hence, for some of these variants, lack of transport activity was not always due to lack of cell surface delivery.


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Fig. 7.   Immunofluorescence studies in X. laevis oocytes expressing NKCC2 variants: saNKCC2A (A), saNKCC2AF+8 (C), and saNKCC2AFno8 (D). Variants were localized in X. laevis oocytes using anti-P-NKCC antibody. Immunofluorescence micrographs are from a representative membrane section among 5 oocytes; exposure times were equal. B: oocytes injected with H2O as control.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The saNKCC2 is a member of the Na-coupled CCCs (Na-CCCs), which constitute one of four branches within the CCC family (Fig. 1); the two other members in the Na-CCC group include NKCC1 (8, 17, 41, 51) and the kidney-specific NCC (8, 9). The second branch consists of the K-coupled CCCs (K-CCC), and the third and fourth branches consist of various CCCs, the substrate(s) of which is unknown (x-CCCs). In the third branch, one of the x-CCCs (also called CIP1) has been found to interact with the NKCC1 (4, 23).

Characterization of an ancient NKCC2 has allowed us to gain important insight into phylogenetic relationships among NKCCs. For instance, Fig. 1A shows that evolutionary distances between saNKCC2 and huNKCC2 are short and similar to those between saNKCC1 and huNKCC1; this pattern is consistent with tissue-specific function for the NKCC2s, which are kidney specific, and the NKCC1s, which are often found in epithelial tissues (47). Figure 1A also shows that the branching point before the two NKCC groups comes close to that of the saNKCC2 or saNKCC1, suggesting that the NKCC2s appeared during the evolution of vertebrates.

The cloning of saNKCC2 makes it possible to determine nonsynonymous-to-synonymous divergence3 (NS/S) ratios (53) over long distances and to identify regions of functional importance (low NS/S ratio) as well as regions that have experienced adaptive substitutions (high NS/S ratio). In this regard, the tm2 region is of particular interest, because it is encoded by alternatively spliced exons (17, 39) that are involved in ion transport (6, 7, 11, 12, 24, 26, 43) and because the second halves of these exons, which encode the connecting loop following the tm2 region (CS2), share higher homology between themselves (and have lower NS/S ratios) than the first halves, which encode the tm2 region per se. These results suggest that CS2 is a key functional domain, whereas the tm2 region is a more adaptable domain. Hence, it is tempting to postulate that true ion-binding residues are found in CS2 and the tm2 region but that affinity-modifying residues, which are presumably species specific, are found mainly in the tm2 region.

We have identified several saNKCC2 splice variants besides A and F. One such variant contains the A and F exons in tandem (saNKCC2AF), as previously reported in mammals (17, 39). Payne and Forbush (39) suggested that this form was an artifact of splicing, because 1 of 32 cDNAs was found to contain both exons and because hydropathy plot models predicted a disrupted structure. In S. acanthias, we found that the AF variant represented ~30% of the NKCC2 transcripts. Moreover, this variant was delivered to the cell surface when expressed in oocytes (Fig. 7). Despite these findings, however, our efforts to achieve functional expression of saNKCC2F were not successful.

Another variant that represented a nonnegligible percentage of saNKCC2 transcripts but had a hydropathy plot model that also predicted a disrupted structure was the saNKCC2AFno8. As for saNKCC2AF+8, it was not possible to obtain functional expression of the no8 variant. In addition, immunofluorescence studies showed that saNKCC2AFno8 was not able to reach the oocyte surface, suggesting that loss of cassette 8 may in fact correspond to an artifact. Replacing the AF exon of the no8 variant by the A or F exon also resulted in inactive transporters.

Recent studies have demonstrated dominant-negative effects of truncated NKCC2 splice variants on NKCC2 (44) as well as of inactive CCCs (mutated NKCC1 and huCIP1) on NKCC1 (4, 21, 23, 25; Darman et al., unpublished data). Similarly, we have found that during the course of the present studies, the AF variant has a dominant-negative effect on saNKCC2A (results not shown). All of these findings suggest that the CCCs are able to form homooligomers at cell surfaces and that conserved domains mediate the interaction. More definitive evidence for dimerization has been obtained recently by Ichinose et al. (19) for the NCC and by Moore-Hoon and Turner (35) for the NKCC1.

Because the NKCC2s are able to form homodimers in vivo, interactions could also occur between different variants, e.g., between A and F, which partly colocalize in the rabbit kidney, or between AF and A or AF and F. As a result, the NKCC2 could presumably exhibit greater functional diversity along the TAL. In the medulla, for instance, dominant-negative effects of inactive NKCC2 variants could influence transport activity. Such a role for the NKCC2AF would be a good reason for high expression levels of the variant in the shark kidney.

Several transport systems are nonfunctional or "modestly" functional, unless they are organized in multimeric structures (3) composed of identical (30), homologous (37), or different subunits (3). Even if various CCCs can interact with one another, it is not known whether homomeric or heteromeric assembly is required for the normal operation of the carriers. Here, coinjection studies have shown that the AF or no8 variants were not inactive because such assembly (AF + no8, AF + A or F, and no8 + A or F) was necessary. These studies revealed that all inactive variants exerted dominant-negative effects on the A or F variants (results not shown).

A significant result in this report is the absence of the "B" exon in the saNKCC2 gene. In mammals, this variant is expressed in the apical membrane of macula densa cells, as evidenced convincingly by functional and localization studies (20, 29, 32, 39, 52); at this site, sensing of luminal salt concentration by NKCC2B is probably crucial for the TGF mechanism (48). During the evolution of bony fishes, it is not clear when this mechanism appeared, although a rudimentary juxtaglomerular apparatus is present in the elasmobranch kidney (31, 38). The NKCC2B could thus represent a phylogenetic marker of mature TAL function in vertebrates, and its presence correlates with the presence of functional macula densa cells.

Homology scores between the alternatively spliced exons suggest that these sequences evolved as a result of exon duplication. Because the shark kidney expresses only the A and F forms and because the B exon is closer to A than it is to F (Fig. 3), these sequences may have appeared in the following order: F right-arrow A right-arrow B. On the basis of recent studies by Giménez et al. (12) showing that rbNKCC2F exhibits ion affinities that are lower than those of rbNKCC2B, a possible link can be made between the evolution from F to B and the passage of vertebrates from higher- to lower-salt environments.

In the oocyte expression system, the time course of activation of saNKCC2 after preincubation in low-Cl medium was surprisingly rapid (Fig. 6). Because these cells have low surface-to-volume ratios, a change in extracellular Cl will bring about a change in intracellular Cl over several hours (46). Thus the rapid rates of activation observed in our studies (t1/2 ~30 min) support a model in which the NKCC2 is a primary site of regulation by Cl, although they do not rule out a model in which change in intracellular Cl affects cotransporter activity through kinase-dependent mechanisms (33, 44). The rapid rates of activation also suggest that a Cl-sensitive site resides in the extracellular domains of NKCC2.

In this report, we also found that the saNKCC2A and saNKCC2F were more active in hypertonic or low-Cl media, consistent with the usual behavior of NKCCs (21, 22, 24, 26, 33, 51). However, saNKCC2F was more sensitive to a change in extracellular osmolality than to a change in extracellular Cl, whereas saNKCC2A was equally sensitive to both stimulating maneuvers. These results suggest that the tm2 region could also play a role in regulation of cotransporter activity.

In conclusion, the cloning and the functional characterization of the S. acanthias NKCC2 have permitted several significant observations. For instance, the appearance of NKCC2B during evolution may correlate with that of the TGF mechanism. In this work, we have also found that the NKCC2 itself may represent an unexpectedly important site of regulation. Further studies are needed to determine the mechanisms by which changes in extracellular Cl (and osmolality) bring about changes in NKCC activity.


    ACKNOWLEDGEMENTS

We thank Claude Villeneuve for superb technical assistance and Michael Cukan for carrying out the initial PCR experiments. Harvesting of S. acanthias kidneys and extraction of poly(A) RNA were performed at the Mount Desert Island Biological Laboratory (Salsbury Cove, ME).


    FOOTNOTES

This study was supported by grants from the Kidney Foundation of Canada, Canadian Institute of Health and Research Grant MT-15405, and National Institutes of Health Grants DK-17433 and ES-3828. P. Isenring is a Canadian Institute of Health and Research Clinician Scientist II.

Preliminary reports of this work have been presented in abstract form (6, 7, 11).

Accession numbers for the spliced variants and saG3.4 are AF521915 for saNKCC2A, AF521917 for saNKCC2F, AF521913 for saNKCC2AF, AF521912 for saNKCC2AFno8, AF521914 for saNKCC2Ano8, AF521916 for saNKCC2Fno8, and AF521976 for saG3.4.

1 It is unlikely that a PCR product from a putative S. acanthias B variant would have had an AccI or an AflIII site; both restriction sites occur in a region where the A, F, and B sequences are divergent in rbNKCC2 and where each of the individual exons is highly conserved.

2 On the other hand, a huNKCC1-rbNKCC2A chimera (hu1rb2A0.7), in which the coding region of rbNKCC2A is replaced with the first 104 NH2-terminal amino acids of the corresponding region in hNKCC1, was active in HEK-293 cells.

3 Nonsynonymous substitutions correspond to amino acid replacements and synonymous substitutions to silent mutations. Nonsynonymous divergence is defined as the number of amino acid replacements per site relative to the total number of sites among homologous sequences and synonymous divergence as the number of silent mutations per site relative to the total number of sites.

Address for reprint requests and other correspondence: P. Isenring, L'Hôtel-Dieu de Québec Research Center, 10 Rue McMahon (Rm. 3852), Quebec, Canada G1R 2J6 (E-mail: paul.isenring{at}crhdq.ulaval.ca).

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

June 4, 2002;10.1152/ajprenal.00107.2002

Received 19 March 2002; accepted in final form 3 June 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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