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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 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.
|
|
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
[-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. laevisExpression 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.
|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
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).
|
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).
|
|
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.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 A
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Beck, PJ,
Orlean P,
Albright C,
Robbins PW,
Gething MJ,
and
Sambrook JF.
The Saccharomyces cerevisiae DPM1 gene encoding dolichol-phosphate-mannose synthase is able to complement a glycosylation-defective mammalian cell line.
Mol Cell Biol
10:
4612-4622,
1990[ISI][Medline].
2.
Biemesderfer, D,
Payne JA,
Lytle CY,
and
Forbush B.
Immunocytochemical studies of the Na-K-Cl cotransporter of shark kidney.
Am J Physiol Renal Fluid Electrolyte Physiol
270:
F927-F936,
1996
3.
Canessa, CM,
Schild L,
Buell G,
Thorens B,
Gautschi I,
Horisberger JD,
and
Rossier BC.
Amiloride-sensitive epithelial Na channel is made of three homologous subunits.
Nature
367:
463-467,
1994[ISI][Medline].
4.
Caron, L,
Rousseau F,
Gagnon E,
and
Isenring P.
Cloning and functional characterization of a cation-Cl cotransporter-interacting protein.
J Biol Chem
275:
32027-32036,
2000
5.
Darman, RB,
Flemmer A,
and
Forbush B.
Modulation of ion transport by direct targeting of protein phosphatase type 1 to the Na-K-Cl cotransporter.
J Biol Chem
276:
34359-34362,
2001
6.
Gagnon, E,
Caron L,
Forbush B,
and
Isenring P.
Activation of NKCC2 splice variants expressed in Xenopus laevis oocytes (Abstract).
J Am Soc Nephrol
10:
32A,
1999.
7.
Gagnon, E,
Caron L,
and
Isenring P.
Functional characterization of the kidney-specific Na-K-2Cl cotransporter from the shark Squalus acanthias (Abstract).
J Am Soc Nephrol
11:
28A,
2000.
8.
Gamba, G,
Miyanoshita A,
Lombardi M,
Lytton J,
Lee WS,
Hediger MA,
and
Hebert SC.
Molecular cloning, primary structure, and characterization of two members of the mammalian electroneutral sodium-(potassium)-chloride cotransporter family expressed in kidney.
J Biol Chem
269:
17713-17722,
1994
9.
Gamba, G,
Saltzberg SN,
Lombardi M,
Miyanoshita A,
Lytton J,
Hediger MA,
Brenner BM,
and
Hebert SC.
Primary structure and functional expression of a cDNA encoding the thiazide-sensitive, electroneutral sodium-chloride cotransporter.
Proc Natl Acad Sci USA
90:
2749-2753,
1993[Abstract].
10.
Gillen, CM,
Brill S,
Payne JA,
and
Forbush B.
Molecular cloning and functional expression of the K-Cl cotransporter from rabbit, rat, and human. A new member of the cation-chloride cotransporter family.
J Biol Chem
271:
16237-16244,
1996
11.
Giménez, I,
Isenring P,
and
Forbush B.
Alternative splicing introduces differences in ion affinities between the cortical and the medullar isoforms of the renal Na-K-Cl cotransporter (Abstract).
J Am Soc Nephrol
10:
32A,
1999.
12.
Giménez, I,
Isenring P,
and
Forbush B.
Spatially distributed alternative splice variants of the renal Na-K-Cl cotransporter exhibit dramatically different affinities for the transported ions.
J Biol Chem
277:
8767-8770,
2002
13.
Greger, R.
Ion transport mechanisms in thick ascending limb of Henle's loop of mammalian nephron.
Physiol Rev
65:
760-797,
1985
14.
Greger, R,
Heitzmann D,
Hug MJ,
Hoffmann EK,
and
Bleich M.
The Na-2Cl-K cotransporter in the rectal gland of Squalus acanthias is activated by cell shrinkage.
Pflügers Arch
438:
165-176,
1999[ISI][Medline].
15.
Haas, M,
and
Forbush B.
The Na-K-Cl cotransporters.
J Bioenerg Biomembr
30:
161-172,
1998[ISI][Medline].
16.
Haas, M,
and
Forbush B.
The Na-K-Cl cotransporter of secretory epithelia.
Annu Rev Physiol
62:
515-534,
2000[ISI][Medline].
17.
Hebert, SC,
and
Gamba G.
Molecular cloning and characterization of the renal diuretic-sensitive electroneutral sodium-(potassium)-chloride cotransporters.
Clin Invest
72:
692-694,
1994[ISI][Medline].
18.
Herrera, VL,
Lopez LV,
and
Ruiz-Opazo N.
1-Na,K-ATPase and Na,K,2Cl-cotransporter/D3mit3 loci interact to increase susceptibility to salt-sensitive hypertension in Dahl S (HSD) rats.
Mol Med
7:
125-134,
2001[ISI][Medline].
19.
Ichinose, M,
Hall AE,
Cheng S,
Xu JZ,
and
Hebert SC.
Novel structure of the flounder thiazide-sensitive Na-Cl cotransporter (flTSC) in the flounder urinary bladder (Abstract).
J Am Soc Nephrol
10:
34A,
1999.
20.
Igarashi, P,
Vanden Heuvel GB,
Payne JA,
and
Forbush B.
Cloning, embryonic expression, and alternative splicing of a murine kidney-specific Na-K-Cl cotransporter.
Am J Physiol Renal Fluid Electrolyte Physiol
269:
F405-F418,
1995
21.
Isenring, P,
and
Forbush B.
Ion and bumetanide binding by the Na-K-Cl cotransporter: importance of transmembrane domain.
J Biol Chem
272:
24556-24562,
1997
22.
Isenring, P,
and
Forbush B.
Ion transport and ligand binding by the Na-K-Cl cotransporter, structure-function studies.
Comp Biochem Physiol
130:
487-497,
2001[ISI].
23.
Isenring, P,
Gagnon E,
and
Caron L.
Cloning and functional characterization of a cation-Cl cotransporter-interacting protein (Abstract).
J Am Soc Nephrol
11:
30A,
2000.
24.
Isenring, P,
Jacoby SC,
Chang J,
and
Forbush B.
Mutagenic mapping of the Na-K-Cl cotransporter for domains involved in ion transport and bumetanide binding.
J Gen Physiol
112:
549-558,
1998
25.
Isenring, P,
Jacoby SC,
and
Forbush B.
Comparison of Na-K-Cl cotransporters: NKCC1, NKCC2, and the HEK cell Na-K-Cl cotransporter.
J Biol Chem
273:
11295-11301,
1998
26.
Isenring, P,
Jacoby SC,
and
Forbush B.
The role of transmembrane domain 2 in cation transport by the Na-K-Cl cotransporter.
Proc Natl Acad Sci USA
95:
7179-7184,
1998
27.
Jacoby, SC,
Gagnon E,
Caron L,
Chang J,
and
Isenring P.
Inhibition of Na-K-2Cl cotransport by mercury.
Am J Physiol Cell Physiol
277:
C684-C692,
1999
28.
Ji, HL,
Fuller CM,
and
Benos DJ.
Osmotic pressure regulates -rENaC expressed in Xenopus oocytes.
Am J Physiol Cell Physiol
275:
C1182-C1190,
1998
29.
Kaplan, MR,
Plotkin MD,
Lee WS,
Xu ZC,
Lytton J,
and
Hebert SC.
Apical localization of the Na-K-Cl cotransporter, rBSC1, on rat thick ascending limbs.
Kidney Int
49:
40-47,
1996[ISI][Medline].
30.
Kreusch, A,
Pfaffinger PJ,
Stevens CF,
and
Choe S.
Crystal structure of the tetramerization domain of the Shaker potassium channel.
Nature
392:
945-948,
1998[ISI][Medline].
31.
Lacy, ER,
and
Reale E.
The presence of a juxtaglomerular apparatus in elasmobranch fish.
Anat Embryol (Berl)
182:
249-262,
1990[ISI][Medline].
32.
Lapointe, JY,
Bell PD,
and
Cardinal J.
Direct evidence for apical Na:2Cl:K cotransport in macula densa cells.
Am J Physiol Renal Fluid Electrolyte Physiol
258:
F1466-F1469,
1990
33.
Lytle, C,
and
Forbush B.
Regulatory phosphorylation of the secretory Na-K-Cl cotransporter: modulation by cytoplasmic Cl.
Am J Physiol Cell Physiol
270:
C437-C448,
1996
34.
Lytle, CY,
Xu JC,
Biemesderfer D,
and
Forbush B.
Distribution and diversity of Na-K-Cl cotransport proteins: a study with monoclonal antibodies.
Am J Physiol Cell Physiol
269:
C1496-C1505,
1995
35.
Moore-Hoon, ML,
and
Turner RJ.
The structural unit of the secretory Na-K-2Cl cotransporter (NKCC1) is a homodimer.
Biochemistry
39:
3718-3724,
2000[ISI][Medline].
36.
Mount, DB,
Mercado A,
Song L,
Xu J,
George AL,
Delpire E,
and
Gamba G.
Cloning and characterization of KCC3 and KCC4, new members of the cation-chloride cotransporter gene family.
J Biol Chem
274:
16355-16362,
1999
37.
Neely, JD,
Christensen BM,
Nielsen S,
and
Agre P.
Heterotetrameric composition of aquaporin-4 water channels.
Biochemistry
38:
11156-11163,
1999[ISI][Medline].
38.
Nishimura, H,
and
Bailey JR.
Intrarenal renin-angiotensin system in primitive vertebrates.
Kidney Int
12:
S185-S192,
1982.
39.
Payne, JA,
and
Forbush B.
Alternatively spliced isoforms of the putative renal Na-K-Cl cotransporter are differentially distributed within the rabbit kidney.
Proc Natl Acad Sci USA
91:
4544-4548,
1994[Abstract].
40.
Payne, JA,
Stevenson TJ,
and
Donaldson LF.
Molecular characterization of a putative K-Cl cotransporter in rat brain. A neuronal-specific isoform.
J Biol Chem
271:
16245-16252,
1996
41.
Payne, JA,
Xu JC,
Haas M,
Lytle CY,
Ward D,
and
Forbush B.
Primary structure, functional expression, and chromosomal localization of the bumetanide-sensitive Na-K-Cl cotransporter in human colon.
J Biol Chem
270:
17977-17985,
1995
42.
Plata, C,
Meade P,
Hall A,
Welch RC,
Vazquez N,
Hebert SC,
and
Gamba G.
Alternatively spliced isoform of apical Na-K-Cl cotransporter gene encodes a furosemide-sensitive Na-Cl cotransporter.
Am J Physiol Renal Physiol
280:
F574-F582,
2001
43.
Plata, C,
Meade P,
Vazquez N,
Hebert SC,
and
Gamba G.
Functional properties of the apical Na-K-2Cl cotransporter isoforms.
J Biol Chem
277:
11004-11012,
2002
44.
Plata, C,
Mount DB,
Rubio V,
Hebert SC,
and
Gamba GG.
Isoforms of the Na-K-2Cl cotransporter in murine TAL. II. Functional characterization and activation by cAMP.
Am J Physiol Renal Physiol
276:
F359-F366,
1999
45.
Race, JE,
Makhlouf FN,
Logue PJ,
Wilson FH,
Dunham PB,
and
Holtzman EJ.
Molecular cloning and functional characterization of KCC3, a new K-Cl cotransporter.
Am J Physiol Cell Physiol
277:
C1210-C1219,
1999
46.
Reifarth, FW,
Amashesh S,
Clauss W,
and
Weber W.
The Ca-inactivated Cl channel at work: selectivity, blocker kinetics and transport visualization.
J Membr Biol
155:
95-104,
1997[ISI][Medline].
47.
Schmidt, TR,
Goodman M,
and
Grossman LI.
Molecular evolution of the COX7A gene family in primates.
Mol Biol Evol
16:
619-626,
1999[Abstract].
48.
Schnermann, J.
Juxtaglomerular cell complex in the regulation of renal salt excretion.
Am J Physiol Regul Integr Comp Physiol
274:
R263-R279,
1998
49.
Shayakul, C,
Tsukaguchi H,
Berger UV,
and
Hediger MA.
Molecular characterization of a novel urea transporter from kidney inner medullary collecting ducts.
Am J Physiol Renal Physiol
280:
F487-F494,
2001
50.
Warth, R,
Bleich M,
Thiele I,
Lang F,
and
Greger R.
Regulation of the Na-2Cl-K cotransporter in in vitro perfused rectal gland tubules of Squalus acanthias.
Pflügers Arch
436:
521-528,
1998[ISI][Medline].
51.
Xu, JC,
Lytle C,
Zhu TT,
Payne JA,
Benz E,
and
Forbush B.
Molecular cloning and functional expression of the bumetanide-sensitive Na-K-Cl cotransporter.
Proc Natl Acad Sci USA
91:
2201-2205,
1994[Abstract].
52.
Yang, T,
Huang YG,
Singh I,
Schnermann J,
and
Briggs JP.
Localization of bumetanide- and thiazide-sensitive Na-K-Cl cotransporters along the rat nephron.
Am J Physiol Renal Fluid Electrolyte Physiol
271:
F931-F932,
1996
53.
Yang, Z,
and
Bielawski JP.
Statistical methods for detecting molecular adaptation.
Trends Ecol Evol
15:
496-506,
2000[ISI][Medline].