1 Groupe de Recherche en Néphrologie, Department of Medicine, Faculty of Medicine, Laval University, Quebec, Canada G1R 2J6; and 2 Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510
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ABSTRACT |
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In the shark (sa), two variants of the renal Na-K-Cl cotransporter (saNKCC2A and saNKCC2F) are produced by alternative splicing of the second transmembrane domain (tm2). In mammals, these splice variants, as well as a third variant (NKCC2B), are spatially distributed along the thick ascending limb of Henle and exhibit divergent kinetic behaviors. To test whether different tm2 in saNKCC2 are also associated with different kinetic phenotypes, we examined the ion dependence of 86Rb influx for shark and rabbit splice variants expressed in Xenopus laevis oocytes. We found that, in both species, A forms have higher cation affinities than F forms. In regard to Cl affinity, however, the A-F difference was more pronounced in rabbit, and the relationship between transport activity and Cl concentration was not always sigmoidal. These results show that the tm2 of saNKCC2 is, as in rabbit, important for Cl transport, and they suggest that the ability of the distal NKCC2-expressing segment to extract Cl from the luminal fluid differs among species. We have also found that the renal NKCC2 of distant vertebrates share similar affinities for cations. This finding points to the existence of highly conserved residues that mediate the kinetic behavior of the NKCC2 splice variants.
cation-chloride cotransporter; renal isoform; splice variant; kidney; NKCC1; NKCC2; thick ascending loop of Henle; kinetics; ion transport
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INTRODUCTION |
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THE SODIUM-POTASSIUM-CHLORIDE cotransporter is an integral membrane protein that facilitates the cellular influx of Na, K, and Cl. Two Na-K-Cl cotransporter genes have been identified, Slc12a2 and Slc12a1, which encode NKCC1 (3, 24, 28) and NKCC2 (23), respectively. NKCC1 is expressed in numerous cell types (9, 22), whereas NKCC2 is expressed exclusively in the kidney (23).
In mammals, the second transmembrane domain (tm2) of NKCC2 is encoded by different exons that are alternatively spliced, leading to the formation of three splice variants: B, A, and F (13, 23, 29). Northern blot analyses of kidney sections (23), in situ hybridizations (13, 23), and immunolocalization studies (19, 29) have shown that these splice variants are differentially distributed along the thick ascending limb of Henle (TAL) as follows: B in the cortex, A in the cortex and the outer stripe of the outer medulla, and F in the inner stripe of the outer medulla.
We recently cloned Squalus acanthias NKCC2 and demonstrated that, in this species, tm2 is also encoded by the A and/or F exon (5); surprisingly, we were not able to identify the B variant and found that this was due to the lack of B exon in the gene (5). The distribution of the splice variants in shark tubules has not been determined; however, immunolocalization studies of kidney sections have allowed detection of NKCCs along the apical margins of cells lining the proximal and distal tubules of the lateral bundle zone (1). Previous microperfusion studies suggested that the latter structure was able to reabsorb Cl by a furosemide-sensitive mechanism (4, 10).
The stoichiometry of Na-K-Cl cotransporters is generally presumed to be 1:1:2; accordingly, these carriers should promote intracellular accumulation of ions without net movement of charge (9). For the squid NKCC1, however, as well as for the other cation-Cl cotransporters (CCCs), stoichiometries do not appear to involve two Cl-binding sites (9, 18, 26), but they are believed to be 3 cations: 3 Cl (squid NKCC1) and 1 cation:1 Cl [Na-Cl cotransporter (NCC) and K-Cl cotransporter (KCC)]. Hence, transport coupling through CCCs may be variable despite high levels of homology shared by the carriers within the family. However, none of these different stoichiometries are predicted to generate electrogenic ion movement.
The NKCC2 plays a central role in TAL physiology. In the medullary portion of this segment, for example, NKCC2 mediates the apical entry step of Na-K(NH4)-Cl reabsorption (8, 20). This movement of ions leads to bulk NaCl reabsorption and contributes to high interstitial osmolalities, an essential component of the urine concentration mechanism. NKCC2 in this region also promotes NH4 transport, thus contributing to the process of renal acidification (6). In the distal TAL, NKCC2 plays a role that is probably equally important by taking part in tubuloglomerular feedback (27).
Structure-function studies have demonstrated that variant residues in tm2 of NKCC1 confer differences in cation affinities among species (14-16), and kinetic analyses of NKCC2 splice variants from two lagomorph species have confirmed the importance of this transmembrane domain in specifying cation and anion affinities (7, 17, 25). These analyses have also shown that the NKCC2 splice variants were individually specialized in their transport characteristics along the TAL (7, 25).
In this work, we have examined the transport kinetics of the A or F form in the shark and the rabbit using the Xenopus laevis oocyte expression system. We found that functional specialization of NKCC2 through alternative splicing may represent a common feature during vertebrate history. We have also confirmed the importance of tm2 in ion translocation by the NKCCs and have identified novel residues that may be involved in the process.
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MATERIALS AND METHODS |
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cDNA construction and vectors. The cDNAs have been described elsewhere (5, 7, 23). Briefly, constructs were engineered in pBluescript SK using full-length NKCC2s as templates [shark (sa) NKCC2AF1 and rabbit (rb) NKCC2A]. The tm2 of these templates (between SphI and PflmI in shark and between SphI and BglII in rabbit) was replaced by the A or F splice variant (shark) or the B or F splice variant (rabbit). Five constructs in pBluescript SK were thus generated: saNKCC2A (saA), saNKCC2F (saF), rbNKCC2B (rbB), rbNKCC2A (rbA), and rbNKCC2F (rbF).
Expression in the X. laevis oocyte.
For functional studies, cDNAs were transferred as EcoRI
inserts in the Pol1 vector; the latter is a modified pGEM vector that contains a T7 promoter, the X. laevis -globin
untranslated regions, and a poly(A) tract (2).
Subsequently, the cDNA-Pol1 constructs were linearized and in vitro
transcribed with T7 RNA polymerase using the mMESSAGE mMACHINE T7 kit (Ambion).
Kinetic studies.
Ion transport rates were determined by 86Rb influx
measurements at ~22°C using various media (Table
1). All these media are derived from a
basic solution that is optimized to elicit cotransporter activity. In
the basic medium, for example, the concentration of K + Rb is 5 mM, close to the Michaelis-Menten constant for Rb
[Km(Rb)] of various NKCCs expressed in
X. laevis oocytes, whereas the concentration of K + Rb in Barth's medium is 3 mM (Table 1).
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RESULTS |
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Functional expression of the NKCC2s.
When expressed in X. laevis oocytes (Fig.
1), each of the wild-type NKCC2 (rbB,
rbA, saA, rbF, and saF) exhibited bumetanide-sensitive 86Rb
influx that was severalfold above that of the endogenous cotransport activity (14-, 16-, 8-, 9-, and 9-fold, respectively). For these experiments, all flux measurements were obtained after hyperosmolar preincubation. Although comparisons of absolute flux rates in heterologous systems must be made cautiously, for example, differences could be accounted for by variation in surface expression, it is
interesting that rbB and rbA tended to exhibit higher transport capacities than rbF, saA, and saF.
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Kinetics of cation translocation.
To determine whether variant residues in tm2 of
elasmobranch NKCC2s convey differences in cation transport [as shown
for rat and rabbit (7, 25)], we measured the dependence
of 86Rb influx on Na and K concentrations for saA and saF
and compared results with those obtained in simultaneous experiments
for rabbit orthologs. Data are illustrated in Fig.
2, A-J, fit by a model of
Na or K binding at a single site; the closeness of the fit for each
condition (expressed as SE of the estimate) was <0.07. Km values derived from these analyses are
depicted in Fig. 3, A and
B.
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Kinetics of Cl transport. Figure 2, K-O, presents results for the Cl dependence of 86Rb influx. The data for rbA, rbF, and saF were fit with the Hill equation using a coefficient of 2. For rbB and saA, the data were poorly fit with this equation; a model of Cl binding at a single site improved the closeness (SE of the estimate <0.07 with a 1-site model vs. >0.10 with a 2-site model). Km values derived from these analyses are depicted in Fig. 3C.
The data show that, for both species, variant residues in tm2 of NKCC2 also convey differences in Cl transport kinetics. As shown above for cation affinities, Cl affinities reported in Fig. 3C are similar to those published recently for the lagomorphs rat and rabbit (7, 25). In this study, however, it appears that, for some of the NKCCs examined, e.g., saA and rbB, variant residues in the alternatively spliced exons are associated with changes in apparent Hill coefficients2; among other possibilities, these changes could be due to an important decrease in the affinity of one of the Cl-binding sites.Structure-function correlations.
To infer which of the NKCC2 variant residues account for distinctive
kinetic behaviors, we analyzed tm2 of several splice variants from different species. We found that when residues at positions 1, 3, 13, 16, and 18 of tm2
were V, I, I, L, and T (as in rbB, rbA, and saA; Fig.
4), carriers had higher affinities for
cations than when these residues were I, V, L, I, and M (as in rbF and
saF). Adding the sequence of the rat NKCC2s to the alignments
(11) showed that variant residues at positions
1 and 3 did not account for divergent kinetic behaviors
among the NKCC2s.
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DISCUSSION |
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We have determined the functional properties of the elasmobranch NKCC2A and F splice variants, which, as in mammals, are produced by alternative splicing of a 96-bp exon encoding tm2. We found that the kinetic properties of the two shark splice variants differed substantially from one another (Figs. 2 and 3). Whereas similar differences are also observed among variants in at least two lagomorph species [rat and rabbit (7, 17, 25)], other characteristics were found to be species specific, e.g., A-F differences in Cl affinity and dependence of transport activity on Cl concentration for some of the splice variants (see below). These results establish that the shark NKCC2 splice variants operate with different substrate affinities, and they also confirm the importance of tm2 in ion translocation.
The characterization of S. acanthias NKCC2 has provided insight into the physiology of the vertebrate nephron in different species. For example, we found that the Km values of each saNKCC2 variant were close to those of the rabbit orthologs. These results suggest that ion concentrations in the lumen of NKCC2-expressing segments are similar between the two species; as discussed in a recent study (7), the Km values of each variant should match the ion concentration of the luminal fluid in which the individual carriers operate. In citing this reference, however, it should be noted that Km values reported here for the rabbit splice variants differ in some measure from those reported by Giménez et al. (7). Micropuncture studies of lateral bundles will therefore be necessary to verify whether the proposed relationship between Km and luminal ion concentration is valid.
Recent kinetic anomalies of rbB have led to the suggestion that certain NKCC2 splice variants could have an altered stoichiometry of transport (7). We have found that, for rbB and saA, the Cl dependence of ion transport was best described with a Hill coefficient of 1. The Hill coefficient is a lower limit for the number of relevant ions involved in transport (16, 21); for these variants, there exists the possibility that only one Cl ion is transported per cycle. Further experiments testing for electrogenicity of transport are necessary to examine this possibility.
The results presented here add to our knowledge of the involvement of tm2 in ion transport. In previous studies on NKCC1, we showed that Cl affinity was specified by variant residues in tm4 and tm7 and not by variant residues in tm2 (15, 16). In this study on NKCC2, we now demonstrate that variant residues in tm2 also influence the kinetics of Cl transport. The approach that was used for the studies on NKCC1 was to exchange segments between shark and human carriers. Hence, important residues that are involved in ion transport, e.g., residues that are the same between shark and human, could have been missed. Taken together, our studies show that the kinetics of Cl transport by the NKCCs are probably specified by at least three different transmembrane regions: tm2, tm4, and tm7.
The characterization of additional NKCC2 splice variants (from S. acanthias) led to the identification of variant residues within tm2 that are associated with specific kinetic phenotypes. For example, carriers with the highest cation affinities (rbB, rbA, saA, and rat NKCC2A) were shown to have I, L, and T in tm2 (positions 13, 16, and 18, respectively), whereas carriers with the lowest cation affinities (rbF, saF, and rat NKCC2F) were shown to have L, I, and M (at the same positions). Residues at these positions were shown to be identical in shark and human NKCC1. For this reason, as mentioned above, their importance in ion translocation could not have been revealed in structure-function studies using a chimera approach (15, 16).
A helical wheel model of tm2 is shown in Fig.
5 to illustrate the position of residues
relative to the lipid bilayer. This model reveals that two of the three
affinity-modifying residues identified in this study have an unexpected
disposition. Indeed, these residues are found within the most
hydrophobic face of the wheel that is presumably in contact with the
lipid bilayer. It is interesting to postulate that such residues may
play a critical role in ion translocation by exerting their influence
from the membrane face of the helix.
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In conclusion, we have found that the A and F forms of S. acanthias NKCC2 operate with different ion affinities. These results indicate that functional specialization through alternative splicing of NKCC2 may correspond to a remarkably conserved feature among a range of vertebrates.
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ACKNOWLEDGEMENTS |
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The authors thank Dr. I. Giménez for reading the manuscript.
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FOOTNOTES |
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This work was supported by a grant 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.
1 NKCC2AF is a splice variant that contains the A and F exons in tandem. Although this variant has been described in several species (5, 13, 25) and was found to be abundantly expressed in S. acanthias, its function is unknown despite extensive functional characterizations (5). It is therefore possible that this variant represents a misprocessed mRNA.
2 In an earlier study, rbB was shown to exhibit peculiar kinetics (7). Although we have not commented on these anomalies, the dependence of rbB on Cl concentration (Fig. 3C of Ref. 7) was also well described by a Hill coefficient that is close to unity.
Address for reprint requests and other correspondence: P. Isenring, L'Hôtel-Dieu de Québec Research Center, 10 Rue McMahon (Rm. 3852), Laval, 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.
First published October 3, 2002;10.1152/ajpcell.00262.2002
Received 4 June 2002; accepted in final form 20 September 2002.
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