Functional Properties of the Apical Na+-K+-2Cl− Cotransporter Isoforms*

The bumetanide-sensitive Na+:K+:2Cl− cotransporter (BSC1) is the major pathway for salt reabsorption in the apical membrane of the mammalian thick ascending limb of Henle. Three isoforms of the cotransporter, known as A, B, and F, exhibit axial expression along the thick ascending limb. We report here a functional comparison of the three isoforms from mouse kidney. When expressed inXenopus oocytes the mBSC1-A isoform showed higher capacity of transport, with no difference in the amount of surface expression. Kinetic characterization revealed divergent affinities for the three cotransported ions. The observed EC50 values for Na+, K+, and Cl− were 5.0 ± 3.9, 0.96 ± 0.16, and 22.2 ± 4.8 mm for mBSC1-A; 3.0 ± 0.6, 0.76 ± 0.07, and 11.6 ± 0.7 mm for mBSC1-B; and 20.6 ± 7.2, 1.54 ± 0.16, and 29.2 ± 2.1 mm for mBSC1-F, respectively. Bumetanide sensitivity was higher in mBSC1-B compared with the mBSC1-A and mBSC1-F isoforms. All three transporters were partially inhibited by hypotonicity but to different extents. The cell swelling-induced inhibition profile was mBSC1-F > mBSC1-B > mBSC1-A. The function of the Na+:K+:2Cl−cotransporter was not affected by extracellular pH or by the addition of metolazone, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), orR(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1-H-indenyl-5-yl)-oxy]acetic acid (DIOA) to the extracellular medium. In contrast, exposure of oocytes to HgCl2 before the uptake period reduced the activity of the cotransporter. The effect of HgCl2 was dose-dependent, and mBSC1-A and mBSC1-B exhibited higher affinity than mBSC1-F. Overall, the functional comparison of the murine apical renal-specific Na+:K+:2Cl−cotransporter isoforms A, B, and F reveals important functional, pharmacological, and kinetic differences, with both physiological and structural implications.

The bumetanide-sensitive Na ϩ :K ϩ :2Cl Ϫ cotransporter is the major salt transport pathway in the apical membrane of the mammalian thick ascending limb of Henle's loop (TALH). 1 The function of this cotransporter in the TALH is critical for salt reabsorption, for the production and maintenance of the countercurrent multiplication mechanism, and is also involved in the regulation of the acid-base and divalent mineral cation metabolism (1). The disruption of the Na ϩ :K ϩ :2Cl Ϫ cotransporter gene in humans (2) and mice (3) produces Bartter's syndrome, an autosomal recessive disease characterized by metabolic alkalosis, hypokalemia, hypercalciuria, and severe volume depletion, accompanied by a reduction in arterial blood pressure. In addition, the Na ϩ :K ϩ :2Cl Ϫ cotransporter protein in the TALH is the main pharmacological target of loop diuretics (4), which are used extensively in the treatment of edematous states.
The primary structure of the kidney-specific, bumetanidesensitive Na ϩ :K ϩ :2Cl Ϫ cotransporter (BSC1 or NKCC2) has been elucidated by cloning cDNA from rat (5), rabbit (6), mouse (7), and human kidney (2). BSC1 belongs to the superfamily of electroneutral cation-coupled chloride cotransporters for which eight genes have been identified (8). Two of these genes encode for Na ϩ :K ϩ :2Cl Ϫ cotransporters: BSC1, a kidney-specific cotransporter expressed only at the apical membrane of the TALH, and BSC2 (also known as NKCC1), a ubiquitously expressed gene at the basolateral membrane of epithelial cells, which is also expressed in several nonepithelial cells. The degree of identity between these proteins is ϳ60%, and in humans, the BSC1 and BSC2 genes are localized in chromosomes 15 and 5, respectively. The murine BSC1 gene gives rise to six alternatively spliced isoforms caused by the combination of two splicing mechanisms. One results from the existence of three mutually exclusive cassette exons of 96 bp named A, B, and F, which encode 31 amino acid residues that are part of the putative transmembrane segment 2 and the connecting segment between transmembrane segments 2 and 3 (6,7). The other splicing mechanism is a polyadenylation signal in the intron between exons 16 and 17 producing a COOH-terminal truncated isoform that lacks the last 327 amino acid residues but contains 55 residues at the end which are not present in the longer isoforms (9). Because the two splicing mechanisms are independent of each other, six isoforms are present in the TALH cells: three isoforms with a long COOH-terminal domain (A, B, and F) and three with a short COOH-terminal domain (A, B, and F) (9,10). * This work was supported in part by Research Grants 97629m from the Mexican Council of Science and Technology (CONACYT) and 75197-553601 from the Howard Hughes Medical Institute (to G. G.) and DK36803 from the National Institutes of Health (to S. C. H. and G. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Supported by scholarship grants from CONACYT and from the Dirección General del Personal Académico of the National University of Mexico.
The functional effect of splicing of the mutually exclusive cassette exons A, B, and F, encoding part of the transmembrane segment 2, is still unknown, but it has been suggested that the exons could affect the transport properties of the cotransporter. Early studies on isolated cortical TALH (cTALH) segments by Burg (13) and medullary TALH (mTALH) segments by Rocha and Kokko (14) indicated that mTALH transports NaCl more rapidly than the cTALH but with greater diluting power in the cTALH (15), suggesting heterogeneity of the transport properties along the TALH. Supporting this possibility, the apparent affinity for Cl Ϫ observed by Greger (16), Hus-Citharel and Morel (17), and Eveloff et al. (18), when cTALH was used as a source of the plasma membrane vesicles, was different from the apparent affinity obtained by Koenig et al. (19) and Burnham et al. (20) when mTALH was used. In this regard, it has been shown that the splicing isoforms A, B, and F exhibit axial distribution along the TALH. The F isoform is absent in the cTALH and present in the mTALH, with higher expression in the inner stripe of the outer medulla. The A isoform is present in both cTALH and mTALH, with higher expression in the outer stripe of the outer medulla, and the B isoform is present only in the cTALH (6,7,21). Thus heterogeneity in the salt transport along the TALH could be caused by the axial distribution of the three isoforms A, B, and F of the Na ϩ :K ϩ :2Cl Ϫ cotransporter. However, the functional characterization of these isoforms has not been addressed.
In the present study, we show a functional characterization of the longer isoforms A, B, and F of the murine Na ϩ :K ϩ :2Cl Ϫ cotransporter using the Xenopus laevis oocytes as an heterologous expression system. Our data revealed significant differences in the affinity for Na ϩ , K ϩ , and Cl Ϫ among isoforms as well as in the sensitivity to bumetanide and response to hypotonicity.

MATERIALS AND METHODS
X. laevis Oocyte Preparation-Adult female X. laevis frogs were obtained from Nasco (Fort Atkinson, MI). Oocytes were harvested by surgery under 0.17% tricaine and incubated for 1 h in the frog Ringer ND96 (in mM: 96 NaCl, 2 KCl, 1.8 CaCl 2 , 1 MgCl, and 5 HEPES/Tris, pH 7.4) in the presence of 2 mg/ml collagenase B. Then, oocytes were washed four times in ND96, defolliculated manually, and incubated overnight in the same medium at 18°C supplemented with 2.5 mM sodium pyruvate and 5 mg/100 ml gentamicin. The next day, stage V-VI oocytes (22) were injected with 50 nl of water or cRNA at a concentration of 0.5 g/l (25 ng of cRNA/oocyte). After injection, oocytes were incubated for 3-4 days in ND96 with sodium pyruvate and gentamicin. The incubation medium was changed every 24 h. The night before the uptake experiments were performed, oocytes were incubated in Cl Ϫ -free ND96 (in mM: 96 sodium isothionate, 2 potassium gluconate, 1.8 calcium gluconate, 1.0 magnesium gluconate, 5 mM HEPES, 2.5 sodium pyruvate, 5 mg% gentamicin, pH 7.4) (23).
In Vitro mBSC1 cRNA Translation-The cloning and preparation of mouse mBSC1 cDNA used in the study have been reported previously (9). In brief, mBSC1-F and mBSC1-A isoforms were cloned by homology from a mouse outer medulla cDNA library, using the flounder thiazidesensitive Na ϩ :Cl Ϫ cotransporter cDNA as a probe (5,9). The short B cassette cDNA was lengthened by PCR and ligated into the BsmI and NsiI sites of the mBSC1-F isoform (9). All of the mBSC1 isoforms used in the present study are inserted in the plasmid pSPORT1 (Invitrogen). To prepare cRNA, each isoform cDNA was linearized at the 3Ј-end using NotI from Roche Molecular Biochemicals, and cRNA was transcribed in vitro, using the T7 RNA polymerase mMESSAGE kit (Ambion). Transcription product integrity was confirmed on agarose gels, and the concentration was determined by absorbance reading at 260 nm (DU 640, Beckman, Fullerton, CA). cRNA was stored frozen in aliquots at Ϫ80°C until used.
To analyze the ion transport kinetics of the Na ϩ :K ϩ :2Cl Ϫ cotransporter isoforms, experiments were performed varying the concentrations of Na ϩ , K ϩ and Cl Ϫ . For Na ϩ kinetics, the extracellular K ϩ and Cl Ϫ concentrations were fixed at 10 and 90 mM, respectively. For K ϩ kinetics, Na ϩ and Cl Ϫ were fixed at 90 mM, and for Cl Ϫ kinetics the Na ϩ and K ϩ concentrations were fixed at 90 and 10 mM, respectively. To maintain osmolarity and ionic strength, N-methyl-D-glucamine was used as an Na ϩ and K ϩ substitute, and gluconate was used as a Cl Ϫ substitute. The transport kinetics for a single ion (Na ϩ , K ϩ , or Cl Ϫ ) was assessed for the three mBSC1 isoforms at the same time, with the same batch of oocytes and solutions. In the same experiment uptake was also measured for each point in water-injected oocytes (data not shown), and the mean values for water groups were subtracted in corresponding mBSC1 groups to analyze only the 86 Rb ϩ uptake because of the injected mBSC1 isoform. Kinetic analysis was performed by estimating the EC 50 values for each ion. The EC 50 values were calculated from log[ion concentration] versus V/V max plots using GraphPad Prism software and an uphill dose-response equation with variable slope (the latter allows the Hill slope to vary from unity). The sensitivity and kinetics for bumetanide were assessed by exposing groups of mBSC1 cRNA-injected oocytes to bumetanide at concentrations varying from 10 Ϫ9 to 10 Ϫ4 M. The desired concentration of the loop diuretic was present in both the incubation and uptake periods. Finally, we also assessed the effect of osmolarity upon the function of mBSC1 isoforms using the following conditions during uptake: hypotonicity of 160, isotonicity of 210, and hypertonicity of 260 mosmol/kg. For these experiments the three mBSC1 isoforms were also analyzed at the same time, and all solutions contained 65 mM NaCl and 5 mM KCl, which resulted in an osmolarity of ϳ 160 mosmol/kg. To prepare the solutions with 210 and 260 mosmol/kg we added 45 and 90 mM sucrose, respectively.
All uptakes were performed at 30°C. At the end of the uptake period, oocytes were washed five times in ice-cold uptake solution without isotope to remove extracellular fluid tracer. After the oocytes were dissolved in 10% SDS, tracer activity was determined for each oocyte by ␤-scintillation counting.
Assessment of mBSC1 Isoform Expression in Oocyte Plasma Membrane-The surface expression of each mBSC1 isoform in the oocyte plasma membrane was measured by fluorescence using enhanced green fluorescent protein (EGFP)-mBSC1 fusion constructs. To make the GFP-mBSC1 fusion constructs, the fragment containing the full-length mBSC1-A cDNA was removed from pSPORT1-BSC1, with the restriction enzymes SalI and NotI, gel isolated and ligated into pEGFP-C1 (CLONTECH, Palo Alto, CA), resulting in the plasmid pEGFP-C1/ BSC1, which contains an in-frame fusion of the mBSC1-A ligated into the COOH terminus of GFP. Then, the cDNA fragment containing the GFP-mBSC1-A was removed from pEGFP-C1/BSC1 by restriction enzyme digestion with AgeI and NotI and ligated into pSPORT1. To obtain GFP-mBSC1-B and GFP-mBSC1-F, the fragment SalI to NsiI of GFP-mBSC1-A, which contains the entire GFP sequence and part of mBSC1 sequence before the second transmembrane domain, was ligated into mBSC1-B and mBSC1-F, which were already in pSPORT1 (9). GFP-mBSC1-A, GFP-mBSC1-B, and GFP-mBSC1-F cRNA was transcribed in vitro and microinjected into X. laevis oocytes (25 ng/oocyte). Water and non-GFP mBSC1-F-injected oocytes were used as control. After 4 days of incubation in regular ND96, oocytes were monitored for GFP fluorescence using a Zeiss laser scanning confocal microscope (objective lens ϫ10, Nikon). Light of excitation wavelength 488 nm and emission 515-565 nm was used to visualize GFP fluorescence. Plasma membrane fluorescence was quantified by determining the pixel intensity around the entire oocyte circumference using SigmaScan Pro image analysis software.
Statistical Analysis-The significance of the differences between groups was tested by one-way analysis of variance with multiple comparison using Bonferroni correction or by the Kruskal-Wallis one-way analysis of variance on ranks with the Dunn method for multiple comparison procedures, as needed. The results are presented as mean Ϯ S.E.

Expression of mBSC1 Isoforms in Xenopus
Oocytes-We and others (5, 24 -26) have shown previously that Xenopus oocytes exhibit an endogenous expression of the bumetanide-sensitive Na ϩ :K ϩ :2Cl Ϫ cotransporter. As shown in Fig. 1, 86 Rb ϩ uptake in H 2 O-injected oocytes was 2,113 Ϯ 346 pmol⅐oocyte Ϫ1 ⅐h Ϫ1 in control conditions and 417 Ϯ 202 pmol⅐oocyte Ϫ1 ⅐h Ϫ1 in the presence of a 10 Ϫ4 M concentration of the loop diuretic bumetanide. Background 86 Rb ϩ uptake was, however, increased by microinjection of X. laevis oocytes with mBSC1-A, mBSC1-B, or mBSC1-F cRNA. The uptake was reduced significantly in all groups in the presence of bumetanide. Thus, to analyze the 86 Rb ϩ uptake induced only by each mBSC1 isoform, in all experiments performed for this study, 86 Rb ϩ uptake was measured simultaneously in water-injected oocytes, and the mean values for the water groups were subtracted in corresponding mBSC1 groups.
As shown in Fig. 1, 86 Rb ϩ uptake in mBSC1-A-injected oocytes was 19,395 Ϯ 1,997 pmol⅐oocyte Ϫ1 ⅐h Ϫ1 , whereas in mBSC1-B oocytes it was 13,229 Ϯ 1,640 pmol⅐oocyte Ϫ1 ⅐h Ϫ1 , and in mBSC1-F it was 12,088 Ϯ 1,561 pmol⅐oocyte Ϫ1 ⅐h Ϫ1 . Thus 86 Rb ϩ uptake in the mBSC1-A isoform is significantly higher than in mBSC1-B and mBSC1-F isoforms (p Ͻ 0.001). The results shown in Fig. 1 are the pooled data from 11 different experiments, using oocytes from different frogs, with an average of 18 oocytes/group in each experiment. The cRNA used was obtained from three different batches, and every time oocytes were injected with the same amount of cRNA (25 ng/ oocyte). The cDNA of the three isoforms used were inserted in the same vector (pSPORT1), contained the same 5Ј-and 3Ј-untranslated regions, and cRNA was transcribed in vitro for the three isoforms simultaneously, using the same T7 RNA polymerase. Thus, differences among isoforms in Fig. 1 are unlikely to be the result of injecting mBSC1-A oocytes with a better quality cRNA, with higher concentration of cRNA/oocyte or that mBSC1-A cRNA was better translated than the other two. Instead, these results suggest that the mBSC1-A isoform exhibits either higher surface expression or higher capacity of transport than the mBSC1-B and mBSC1-F isoforms. To determine whether the differences in functional expression were caused by variation in the surface expression of the Na ϩ :K ϩ : 2Cl Ϫ cotransporter isoforms, X. laevis oocytes injected with GPF-mBSC1-A, GFP-mBSC1-B, or GFP-mBSC1-F cRNA isoforms were analyzed by confocal fluorescence microscopy. Figs. 2, A-D, present a representative picture of oocytes injected with each isoform, and Fig. 2E shows the result of these experiments in which at least 40 oocytes/isoform were evaluated. As shown in Fig. 2E, although numbers were smaller on mBSC1-F-injected oocytes (31,212 Ϯ 4,165; n ϭ 48) than in those injected with mBSC1-A (48,888 Ϯ 8,042; n ϭ 50) or mBSC1-B (43,995 Ϯ 8,495; n ϭ 40), analysis of variance showed no significant differences in surface expression among the three isoforms. Thus, under our experimental conditions it is unlikely that the type of mutually exclusive cassette exon affects the surface expression of the cotransporter in oocytes. This observation supports the hypothesis from Fig. 1 that mBSC1-A might be the isoform with the highest capacity of transport.
Transport Kinetics of mBSC1 Isoforms-The kinetic transport properties for each ion were assessed for the three isoforms simultaneously, in the same batch of injected oocytes. Fig. 3A shows the Na ϩ transport kinetics of each isoform, and panels B, C, and D depict the Hill coefficient plots for Na ϩ in mBSC1-B, mBSC1-A, and mBSC1-F, respectively. The Na ϩ dependence of 86 Rb ϩ uptake was assessed with fixed concentrations of K ϩ and Cl Ϫ at 10 and 96 mM, respectively, with changing concentrations of Na ϩ from 0 to 80 mM. 86 Rb ϩ uptake increased as the Na ϩ concentration was increased until a plateau phase was reached, compatible with Michaelis-Menten behavior. Table I shows the EC 50 and Hill coefficient values. The EC 50 values for Na ϩ were similar between mBSC1-A and mBSC1-B isoforms but different from the values observed for the mBSC1-F isoform. Fig. 4A shows the K ϩ transport kinetics of each isoform, and panels B, C, and D depict the Hill coefficient plots for K ϩ in mBSC1-B, mBSC1-A, and mBSC1-F, respectively. The experiments were performed with fixed concentrations of Na ϩ and Cl Ϫ at 96 mM, with increased concentrations of K ϩ from 0 to 10 mM. The 86 Rb ϩ uptake increased as the K ϩ concentration increased in the extracellular medium until a plateau phase was reached. EC 50 and Hill coefficients are shown in Table I. As with Na ϩ transport kinetics, the EC 50 values observed in mBSC1-A and mBSC1-B were similar, whereas the EC 50 for K ϩ in mBSC1-F isoform was higher. Fig. 5A depicts the Cl Ϫ transport kinetics for each mBSC1 isoform, and panels B, C, and D show the Hill plots for Cl Ϫ . These experiments were carried out with Na ϩ and K ϩ concentrations fixed at 96 and 10 mM, respectively, with increased Cl Ϫ concentrations from 0 to 96 mM. 86 Rb ϩ uptake increased as a function of the Cl Ϫ concentration. The plateau phase was reached in mBSC1-A and mBSC1-B, but not in mBSC1-F. As shown in Table I, the EC 50 value for Cl Ϫ was higher in mBSC1-F than in mBSC1-A or mBSC1-B. Hill coefficients for Na ϩ and K ϩ in the three isoforms were close to unity, whereas Hill coefficients for Cl Ϫ were above unity, consistent with the 1Na ϩ , 1K ϩ , and 2Cl Ϫ stoichiometry. As gesting that affinity for each ion is similar between these two isoforms. In contrast, the EC 50 values for Na ϩ , K ϩ , and Cl Ϫ in mBSC1-F-injected oocytes were higher, suggesting that this is the isoform with the lowest affinity for the cotransported ions.
Kinetics of Bumetanide Inhibition of mBSC1 Isoforms-Bumetanide-induced inhibition of cotransport activity is one of the hallmarks of the Na ϩ :K ϩ :2Cl Ϫ cotransporter. Thus, we analyzed the inhibitory kinetics of bumetanide on mBSC1-A, mBSC1-B, and mBSC1-F transport in oocytes. As shown in Fig.  6, all three isoforms were inhibited by the loop diuretic in a dose-dependent manner. However, the IC 50 for bumetanide inhibition of 86 Rb ϩ uptake was lower in mBSC1-B (600 nM) than in mBSC1-A (2 M) or mBSC1-F (3.4 M). In addition, the percentage of inhibition of the Na ϩ :K ϩ :2Cl Ϫ cotransporter function from 10 Ϫ7 to 10 Ϫ5 M concentration was significantly higher in mBSC1-B than in mBSC1-F and mBSC1-A. Thus, the  3. Kinetic transport analysis for Na ؉ in mBSC1 isoforms. Panel A, Na ϩ -dependent 86 Rb ϩ uptake in X. laevis oocytes injected with mBSC1-A (circles), mBSC1-B (boxes), and mBSC1-F (triangles) cRNA. The experiment was performed with increasing Na ϩ concentrations of 0.5, 1, 2, 3.5, 5, 10, 20, 40, and 80 mM, with the concentrations of K ϩ and Cl Ϫ fixed at 10 and 96 mM, respectively. Lines were fit using the Michaelis-Menten equation. Each point represents the mean Ϯ S.E. of 15 oocytes. Panels B, C, and D show the Hill plots for Na ϩ in mBSC1-B, mBSC1-A, and mBSC1-F, respectively. mBSC1-B isoform exhibited higher affinity for bumetanide than the other two isoforms.
Regulation of mBSC1 Isoforms by Osmolarity-As all members of the electroneutral cation-coupled chloride cotransporter family, the Na ϩ :K ϩ :2Cl Ϫ cotransporter is a cell volume-regulated protein. We have shown before (5) a significant reduction of the rat BSC1-F cotransporter function when oocytes were incubated in hypotonic medium (ϳ160 mosmol/kg) compared with isotonic frog Ringer (ϳ210 mosmol/kg). We also observed in hypotonic medium that the reduction of the endogenously expressed Na ϩ :K ϩ :2Cl Ϫ cotransporter in oocytes was significantly higher than the inhibition observed in rat BSC1-F, suggesting that sensitivity to cell volume might be different among Na ϩ :K ϩ :2Cl Ϫ cotransporter isoforms. Accordingly, we assessed the bumetanide-sensitive 86 Rb ϩ uptake in oocytes injected with mBSC1-A, mBSC1-B, and mBSC1-F cRNA and exposed to an uptake medium containing 65 mM NaCl at three different osmolarities: hypotonic (ϳ 160 mosmol/kg, the osmolarity obtained by the 65 mM NaCl concentration in the uptake medium), isotonic (ϳ 210 mosmol/kg), or hypertonic (ϳ 260 mosmol/ kg) with sucrose added to the 65 mM NaCl uptake medium to adjust osmolarity. Therefore, 86 Rb ϩ uptake was assessed in three osmolar conditions, without differences in extracellular NaCl concentration or ionic strength. The uptake in isotonic medium was taken as 100% activity. As shown in Fig. 7, incubation of oocytes in 260 mosmol/kg resulted in a significant increase in the activity of the endogenously expressed oocyte Na ϩ :K ϩ :2Cl Ϫ , whereas the activity of the mBSC1 isoforms was unchanged. When 86 Rb ϩ uptake was performed in 160 mosmol/ kg, the endogenous oocyte Na ϩ :K ϩ :2Cl Ϫ cotransporter activity was inhibited completely (5.1 Ϯ 1.0% of the function observed in isotonicity), whereas the activity of mBSC1 isoforms was only partially reduced, but to a different extent among the isoforms. Comparing with uptake assessed in isotonicity, the 86 Rb ϩ uptake in 160 mosmol/kg in mBSC1-A was 74 Ϯ 3.3%, in mBSC1-B was 57 Ϯ 3.2%, and in mBSC1-F was 46 Ϯ 2.9% (p Ͻ 0.01). Thus, the cell swelling-induced inhibition profile of the Na ϩ :K ϩ :2Cl Ϫ cotransporter isoforms was mBSC1-F Ͼ mBSC1-B ϾmBSC1-A.  4. Kinetic transport analysis for K ؉ in mBSC1 isoforms. Panel A, K ϩ -dependent 86 Rb ϩ uptake in oocytes injected with mBSC1-A (circles), mBSC1-B (boxes), and mBSC1-F (triangles) cRNA. Uptake was assessed in the presence of increasing K ϩ concentrations of 0.1, 0.25, 0.4, 0.6, 1.0, 2, 5, and 10 mM. For the K ϩ kinetics analysis the Na ϩ and Cl Ϫ concentration was fixed at 96 mM. Lines were fit using the Michaelis-Menten equation. Each point represents the mean Ϯ S.E. of 15 oocytes. Panels B, C, and D show the Hill plots for K ϩ in mBSC1-B, mBSC1-A, and mBSC1-F, respectively. Fig. 8A shows the 86 Rb ϩ uptake in X. laevis oocytes injected with each of the mBSC1 isoforms and exposed to extracellular pH from 6.0 to 8.0. Fig. 8B shows the percentage of bumetanide inhibition of each isoform. Uptake experiments were performed in solutions containing 96 mM NaCl and 10 mM KCl, with pH values of 6.0, 6.5, 7.0, 7.5, and 8.0. Bumetanide was used at 5 ϫ 10 Ϫ7 M. As shown in Fig. 8A, 86 Rb ϩ uptake was  (hatched bars), mBSC1-A (white bars), mBSC1-B (black  bars), and mBSC1-F (gray bars). Uptake was assessed in the absence and presence of 10 Ϫ4 M bumetanide, and the mean value of the bumetanide groups was subtracted in the corresponding control group to show the bumetanide-sensitive portion of the uptake. Oocytes were exposed to uptake media with osmolarities of 160, 210, or 260 mosmol/ kg. * indicates p Ͻ 0.05 versus the uptake in isotonicity. † indicates p Ͻ 0.01 versus all other groups in 160 mosmol/kg. Each point represents the mean Ϯ S.E. of 40 oocytes from two different frogs. similar from 6.0 to 8.0 for each isoform. Thus, we observed no difference in the Na ϩ :K ϩ :2Cl Ϫ cotransporter activity at different pH values. Also, as shown in Fig. 8B, no significant difference was observed in the degree of bumetanide inhibition of each isoform at pH from 6.0 to 8.0. Note, however, that at most of the studied pH values, the degree of inhibition by 5 ϫ 10 Ϫ7 M bumetanide was significantly lower in mBSC1-F isoform, except when uptake was performed at 7.5, suggesting that lower or higher pH magnified the difference in bumetanide sensitivity among mBSC1 isoforms, making mBSC1-B and mBSC1-A more sensitive to the effect of loop diuretics than mBSC1-F.

Effect of pH on rBSC1 Function and Bumetanide Inhibition-
Effect of Inhibitors and Mercury-The electroneutral cationcoupled chloride cotransporters are defined in part by their sensitivity to several diuretics and inhibitors. For instance, thiazide-type diuretics are specific inhibitors of the Na ϩ :Cl Ϫ cotransporter (5), and the alkaloid compound DIOA has been proposed as a specific inhibitor of the K ϩ :Cl Ϫ cotransporter (27). In addition, other drugs such as the stilbene compounds exhibit inhibitory properties upon Cl Ϫ transporters, including the Cl Ϫ -HCO 3 Ϫ exchanger (28), the K ϩ :Cl Ϫ cotransporter (29), and the thiazide-sensitive cotransporter (5). Thus, we assessed the effect of metolazone, DIOA, or DIDS upon 86 Rb ϩ uptake in mBSC1-F-injected oocytes. As shown in Fig. 9, the thiazide-like diuretic metolazone, the alkaloid DIOA, and the stilbene DIDS had no inhibitory properties upon the Na ϩ :K ϩ :2Cl Ϫ cotransporter. As expected, a 10 Ϫ4 M concentration of bumetanide resulted in complete inhibition of the cotransporter activity. In addition to the specific inhibitors, it is well known that many ion transporters are affected by exposure to HgCl 2 . In the electroneutral cotransporter family, Mercado et al. (30) have shown that the X. laevis K ϩ :Cl Ϫ cotransporter in oocytes is activated by HgCl 2 , whereas Jacoby et al. (31) found that the basolateral isoform of the Na ϩ :K ϩ :2Cl Ϫ cotransporter is inhibited by HgCl 2 , and we also have evidence that HgCl 2 reduces the function of the thiazide-sensitive Na ϩ :Cl Ϫ cotransporter (32). As shown in Fig. 9, we also analyzed the effect of 50 M HgCl 2 upon the 86 Rb ϩ uptake induced by mBSC1-F. A significant inhibitory effect of HgCl 2 on the function of the apical Na ϩ :K ϩ :2Cl Ϫ cotransporter was observed. Then, to assess the effects of HgCl 2 on the three isoforms, X. laevis oocytes injected with mBSC1-A, mBSC1-B, or mBSC1-F cRNA were exposed to increased concentrations of extracellular HgCl 2 from 1 to 75 M. Higher concentrations were not used because we have observed a dramatic increase in 86 Rb ϩ uptake in oocytes when HgCl 2 is used at 100 M or above (30). As shown in Fig. 10, the exposure of mBSC1 isoforms to HgCl 2 resulted in significant and dose-dependent inhibition of the cotransporter function. In addition, mBSC1-A and mBSC1-B exhibited a similar pattern of inhibition, whereas the percentage of reduction in the function of mBSC1-F was significantly lower than in the other isoforms. DISCUSSION The gene encoding for the apical Na ϩ :K ϩ :2Cl Ϫ cotransporter in mouse gives rise to six alternatively spliced isoforms that are expressed exclusively in the apical membrane of the TALH (9). On one hand, two isoforms are produced after truncation of the COOH-terminal domain. The longer isoform is made up of 1,095 amino acid residues, and the shorter isoform has 770 residues. On the other hand, three isoforms are produced because of the existence of three 96-bp mutually exclusive cassette exons designated A, B, and F, which encode part of the transmembrane domain 2 and the connecting segment between transmembrane domains 2 and 3 (7). Because this splicing mechanism can be combined with the COOH-terminal domain splicing, then, six isoforms are produced: three with a long COOH-terminal domain and three with a short COOH-termi- nal domain (1). We have shown that the three long COOHterminal domain isoforms encode for the bumetanide-sensitive Na ϩ :K ϩ :2Cl Ϫ cotransporter (11) and that the short isoforms exert a dominant-negative effect upon the Na ϩ :K ϩ :2Cl Ϫ cotransporter which can be abrogated by cAMP (11). In addition, we have also demonstrated that the shorter isoforms work as hypotonically activated, bumetanide-sensitive, K ϩ -independent, Na ϩ :Cl Ϫ cotransporter, which is inhibited by activation of protein kinase A with cAMP (12).
In the present study we have established the major properties of the three long isoforms A, B, and F of the murine apical Na ϩ :K ϩ :2Cl Ϫ cotransporter. The long isoforms mBSC1-A and mBSC1-B exhibit transport kinetic properties for Na ϩ , K ϩ , and Cl Ϫ which are similar between each other but different from the transport kinetic properties observed in the long isoform mBSC1-F. Our data show that this last isoform possesses the lowest affinity for the cotransported ions. In addition, although surface expression of the three isoforms in the oocytes plasma membrane is similar (Fig. 2), 86 Rb ϩ uptake was significantly higher in mBSC1-A-injected oocytes, even after 11 experiments were pooled together (Fig. 1), suggesting that this isoform could have a higher transport capacity. Taking all of these data together, we propose that mBSC1-A is the high affinity, high capacity transporter; mBSC1-B is the high affinity, low capacity isoform; and mBSC1-F is the low affinity, low capacity isoform. These transport kinetics properties are in accordance with the localization of the isoforms along the TALH. It has been shown that mTALH possesses a higher capacity for NaCl transport than cTALH, but cTALH possesses a higher capacity for ion dilution (13)(14)(15). At the beginning of the TALH, ion concentrations in the tubular fluid that comes from the inner medulla are very high; but as TALH reaches the cortex, the concentration of ions is reduced because of the combination of intense salt reabsorption and low water permeability. In fact, at the end of the cTALH the tubular fluid is more diluted than plasma. Accordingly, the mBSC1-A isoform, which exhibits the higher capacity of transport, is present all along TALH, but with higher expression levels in the outer medulla. In addition, mBSC1-F, the isoform with the lower affinity for the cotransported ions, has been localized only in the mTALH, with predominant expression at the inner stripe of the outer medulla where ion concentration is very high (7,21). Thus, the higher capacity of transport in mTALH can be the result of the higher expression of the mBSC1-A cotransporter. In contrast, in cTALH mBSC1-B is the predominant isoform, with some expression of mBSC1-A. These two isoforms exhibit high affinity for the cotransported ions, with EC 50 values for Na ϩ (ϳ3 mM), K ϩ (ϳ 1 mM), and Cl Ϫ (11-20 mM) which are clearly below the concentration of these ions in tubular fluid, allowing the reabsorption of salt to take place, even when tubular fluid is more diluted than plasma. Thus the expression of the high affinity isoforms mBSC1-B and mBSC1-A in cTALH can be the reason behind the greater dilution power of cTALH compared with mTALH. Isenring et al. (33)(34)(35) performed a series of chimera clones and point mutations between the human and shark basolateral isoform of the Na ϩ :K ϩ :2Cl Ϫ cotransporter, known as NKCC1 or BSC2, and concluded that the transmembrane domains important to define kinetic properties are domains 2 and 4 for Na ϩ affinity; 2, 4, and 7 for K ϩ affinity; and only 4 and 7 for Cl Ϫ affinity. Here we show that mutually exclusive cassette exons A, B, and F in mBSC1 are critical for defining the affinity for the three cotransported ions. We cannot verify the role of other membrane spanning domains in ion affinities because, with exception of the exon cassettes, the rest of the mBSC1 isoforms are identical. However, the fact that the only difference among mBSC1-A, mBSC1-B, and mBSC1-F is the exon cassette indicates that in the apical Na ϩ :K ϩ :2Cl Ϫ cotransporter, this is the region that defines differences in affinities for Na ϩ , K ϩ , as well as for Cl Ϫ .
We observed some correlation between the affinity for ions and for bumetanide. mBSC1-F exhibits the lower affinity for ions and also for bumetanide, whereas mBSC1-B behaves as the isoform with the higher affinity for Cl Ϫ and also for bumetanide. In this regard, Isenring and Forbush (36) showed that affinity for Na ϩ , K ϩ , Cl Ϫ and bumetanide of the human basolateral Na ϩ :K ϩ :2Cl Ϫ cotransporter is higher than the shark ortholog, and we have made a similar observation in the thiazide-sensitive Na ϩ :Cl Ϫ cotransporter: the rat cotransporter exhibits higher affinity for Na ϩ , Cl Ϫ , and also for thiazides, than the winter flounder urinary bladder ortholog (23,37), indicating that in members of the electroneutral cotransporter family, the higher affinity for the cotransported ions is accompanied by higher affinity for inhibitors. These observations support the hypothesis that inhibition of the cotransporter activity by bumetanide probably involves competence between ions (particularly Cl Ϫ ) and the loop diuretic for the same site on the protein (38).
In the present study we observed a significant difference in the response to changes in cell volume by mBSC1 isoforms. When oocytes were exposed to variations in extracellular osmolarity, the change in mBSC1 function was different among the three isoforms. During cell swelling, the decrease in cotransporter function was 54% in mBSC1-F, 43% in mBSC1-B, and 26% in mBSC1-A; during cell shrinkage the increase in cotransporter activity was 24, 9, and 1%, respectively. Thus mBSC1-F is the isoform with the highest sensitivity to changes in cell volume. We also observed that endogenous Na ϩ :K ϩ :2Cl Ϫ cotransporter in oocytes exhibited an even higher sensitivity to cell volume because the function of this cotransporter was inhibited by 95% during cell swelling and activated by 44% in hypertonicity. The reduction in cotransporter activity in our experiments was observed by changing the normal osmolarity for oocytes from ϳ 210 to 160 mosmol/kg; i.e. about 25% change. This osmolarity (160 mosmol/kg) is unlikely to be present in mammalian renal medulla. However, similar and even higher percentages of reduction in renal medulla osmolarity can occur as a consequence of water loading. Under these conditions, the interstitial NaCl and urea concentrations drop rapidly, and renal medulla tonicity is reduced; however, because of the high contents of osmolytes, such as betaine, inositol, or sorbitol within the mTALH cells, when extracellular osmolarity is reduced, cells take up water and swell (9). Along the TALH, this phenomenon occurs with more intensity in the inner stripe of the outer medulla, where the mBSC1-F isoform is mainly expressed. Thus, our observation of mBSC1-F as the isoform with the higher sensitivity for changes in cell volume agrees with its proposed localization. The present study, however, does not elucidate the mechanisms by which hypotonicity reduces the function of the mBSC1 isoforms to a different extent.
During the first half of the 20th century, mercurials were used as the first potent diuretic agents (39); they were later discontinued because of their high toxicity and the tendency toward tachyphylaxis, in addition to the concomitant development of better diuretic agents such as loop diuretics and thiazides. The site of action in the nephron was localized at the thick ascending limb and distal nephron, where mercury inhibited net Cl Ϫ reabsorption (40). However, the mechanism of action was never determined. We have observed recently that mercury reduces the function of both the rat and the flounder thiazide-sensitive Na ϩ :Cl Ϫ cotransporter (32). In addition, Jacoby et al. (31) have shown that the basolateral isoform of the Na ϩ :K ϩ :2Cl Ϫ cotransporter can also be inhibited by mercury.
In the present study we show that exposure of X. laevis oocytes to HgCl 2 few minutes before the beginning of the uptake period resulted in a significant and dose-dependent reduction of mBSC1 activity. Thus, the diuretic effect of mercury could be caused by direct inhibition of both the Na ϩ :K ϩ :2Cl Ϫ and the Na ϩ :Cl Ϫ cotransporters located at the apical membrane of the TALH and the distal tubule, respectively. Depicted in Fig. 11 are the amino acid sequences of the mutually exclusive cassette exons from mouse kidney (7,9). Although the exons expand 31 amino acid residues, differences among isoforms are small. There are only three amino acid residues that are completely different in the three isoforms. In addition to these three residues, some amino acids are different in one isoform compared with the other two. For instance, the leucine, isoleucine, methionine, and cysteine marked on mBSC1-F are different in mBSC1-A and mBSC1-B, but these residues are identical in isoforms A and B, suggesting that these four amino acid residues could be responsible for kinetic differences between mBSC1-A and mBSC-B, with mBSC1-F isoforms. Particularly interesting is the presence of one methionine and cysteine in mBSC1-F which could confer different tertiary structure to this isoform.
In summary, our data revealed significant kinetic, pharmacological, and regulatory differences among the isoforms A, B, and F of the murine Na ϩ :K ϩ :2Cl Ϫ cotransporter. Because the only structural variation among these three isoforms is the mutually exclusive cassette exon, some amino acid residues within these exons must be responsible for the observed differences in functional properties. Further studies will be neces-sary to elucidate the role of each different amino acid residue of the exon cassettes upon the functional properties of mBSC1 isoforms shown in the present study.