Functional comparison of renal Na-K-Cl cotransporters between distant species

Édith Gagnon1, Biff Forbush2, Luc Caron1, and Paul Isenring1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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).

Defolliculated stage V-VI oocytes were injected with 25 nl of H2O or ~5-25 ng of cRNA diluted in 25 nl of H2O. Oocytes were tested only 3-5 days after injection to allow full expression of the heterologous protein. During this preliminary period, eggs were maintained at 18°C in Barth's medium; furosemide (125 µM) was also added to the medium to prevent NKCC-injected eggs from becoming increasingly brittle after injection (personal observations); presumably, foreign cotransporters induce large influx of ions that become toxic to eggs over several hours.

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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Table 1.   Composition of flux solutions

Before the functional assays, furosemide was removed with washes in the basic isosmolar solution. Oocytes were then incubated for 1 h in a tracer-free basic solution supplemented with 84 mosM sucrose (final osmolality 284 mosM). Such a maneuver has been shown to activate NKCC1 and NKCC2 in native tissues (9) as well as in various expression systems (2, 5-7, 17, 25). However, not all cotransporters require hyperosmolar preincubation to become fully activated, e.g., the rabbit NKCC2 variants (5), and they probably do not remain stably activated 1 h after the preincubation. For the present studies, nevertheless, all NKCC2-injected eggs were subjected to the same preflux maneuvers.

After the activating maneuvers, eggs were reincubated for 45 min in the basic solution or in various isosmolar solutions containing 1-2 µCi/ml 86Rb and 10 µM ouabain with or without 250 µM bumetanide; high bumetanide concentrations are used to inhibit cotransporter activity throughout the 45-min flux assay. The various isosmolar solutions consist of basic solutions that are modified by varying the concentration of Na (from 0 to 87 mM), Rb (from 0.1 to 20 mM), or Cl (from 0 to 86 mM). In these studies, Na or Rb is replaced with N-methyl glucamine and Cl with gluconate without or without SO4 (Table 1). N-methyl glucamine, gluconate, and SO4 do not appear to be transported by the NKCCs or to influence NKCC-mediated Rb transport (personal observations; 12).

Fluxes were terminated with washes in a basic isosmolar medium containing 250 µM bumetanide and 10 µM ouabain. After the final washes, oocytes were transferred in 96-well plates (1 oocyte/well) and solubilized in 2% SDS. 86Rb was detected by liquid beta scintillation counting using the TopCountNXT microplate counter (Packard).

In each experiment, fluxes among two to six oocytes (usually 4-5 oocytes) were averaged; for studies in which the ion dependence of transport was assessed, these averaged fluxes were normalized to the value measured at the highest ion concentration. Flux values (absolute or normalized) from one to five experiments (usually 3-4 experiments) were subsequently reaveraged to obtain means ± SE. Michaelis-Menten constants (Km) were determined by nonlinear least-square analysis using the Michaelis-Menten equation (for a 1-binding-site model) or the Hill equation with a coefficient of 2 (for a 2-binding-site model). 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. Closeness of fit is expressed as SE on the basis of iterative estimates obtained with SigmaPlot 4.00 for Windows.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   86Rb bumetanide (Bu)-sensitive influx by Xenopus laevis oocytes injected with H2O, rabbit (rb) NKCC2B, rbNKCC2A, or rbNKCC2F, or shark (sa) NKCC2A or saNKCC2F. After a 45-min incubation in hyperosmotic medium, oocytes were assayed for 86Rb influx in a basic medium + 10 µM ouabain. Values are means ± SE of 4-6 oocytes from 1-5 experiments. Composition of media used for these studies is shown in Table 1. Data for saNKCC2s are from Ref. 5.

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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Fig. 2.   Dependence of 86Rb influx on Na, Rb, and Cl concentrations for 5 different NKCC2 cotransporters. When Na concentration was varied from 0 to 87 mM (A-E), Rb and Cl concentrations were 5 and 86 mM, respectively. When Rb concentration was varied from 0.1 to 20 mM (F-J), Na and Cl concentrations were 87 and 86 mM, respectively. When Cl concentration was varied from 0 to 86 mM (K-O), Na and Rb concentrations were 87 and 5 mM, respectively. For some of the NKCCs, an appreciable influx of Rb persists in the absence of Na (A-C). We believe that this residual influx, which is largely bumetanide sensitive and is also seen in NKCC2-transfected HEK-293 cells (17), represents Rb/K exchange. Data in A-O are averages of 2-6 oocytes from 2-4 experiments. For Na and Rb (A-J), points are fit by the Michaelis-Menten equation. For Cl (K-O), points are fit by the Michaelis-Menten equation (rbNKCC2B and saNKCC2A) or by the Hill equation using a Hill coefficient (n) = 2 (rbNKCC2A, rbNKCC2F, and saNKCC2F).



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Fig. 3.   Michaelis-Menten constants (Km) for Na [Km(Na), A], Rb [Km(Rb), B], and Cl [Km(Cl), C] for the 5 cotransporters. Values were obtained by averaging Km values of individual experiments and are presented as means ± SE. For saNKCC2A, Km(Cl) obtained by this calculation was higher than that obtained by a fit of the averaged data shown in Fig. 2M (38 vs. 16 mM). At 16 mM, the A-F difference in Cl affinity would still be much smaller in shark than in rabbit. All other Km values that were calculated by averaging fits of individual experiments were nearly identical to those obtained by fits of averaged data in Fig. 2. * Difference between NKCC2s is statistically nonsignificant. ** Difference between NKCC2s is statistically significant (P < 0.01).

The data fall into two groups: higher-affinity carriers (rbB, rbA, and saA) and lower-affinity carriers (rbF and saF). The data also show that Km(Rb) for a given variant is similar between the two species and that the same principle applies for Km(Na), except saA has slightly higher Na affinity than rbA. The Km values reported here for the rabbit are similar to those reported by us in recent publications (7, 17) and to those reported for another lagomorph, the rat (25).

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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Fig. 4.   Alignments of tm2 of 5 NKCCs. Boxes highlight residues that are identical among the 5 NKCCs; color coding highlights residues that are associated with distinct phenotypes. Position 1 is attributed to the 1st residue of the helix and position 18 to the last residue of the helix.

The analyses described here also led to the identification of variant tm2 residues that were associated with distinctive behaviors in regard to Cl transport. For example, when the residue at position 7 was an alanine (as in rbB and saA), the flux data measured at various substrate concentrations were fit best by a Hill coefficient of 1. On the other hand, when this residue was a serine (as in rbA, rbF, and saF), the flux data were fit best by a Hill coefficient of 2.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 5.   Helical wheel model of the 2nd transmembrane domain (tm2). Wheel was drawn using a 100° angle. Boxed letters indicate candidate affinity-modifying residues identified in this study. Shaded area depicts the most hydrophobic face of the helix. Position (Pn) 1 is attributed to the 1st residue of the helix and position 18 to the last residue of the helix.

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.


    ACKNOWLEDGEMENTS

The authors thank Dr. I. Giménez for reading the manuscript.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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