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• W1966841727 abstract "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 in Xenopus 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), or R(+)-[(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+: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 in Xenopus 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), or R(+)-[(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+:K+:2Cl− cotransporter is the major salt transport pathway in the apical membrane of the mammalian thick ascending limb of Henle's loop (TALH). 1The abbreviations used are: TALHthick ascending limb of Henle's loopcTALHcortical TALHmTALHmedullary TALHBSC1bumetanide-sensitive cotransporter 1 (also known as NKCC2)BSC2bumetanide-sensitive Na+-K+-2Cl− cotransporter 2 (also known as NKCC1)mBSC1mouse BSC1DIDS4,4′-diisothiocyanatostilbene-2,2′-disulfonic acidDIOAR(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1-H-indenyl-5-yl)-oxy]acetic acidGFPgreen fluorescent proteinEGFPenhanced GFP86Rb+tracer rubidium 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.Gamba G. Kidney Int. 1999; 56: 1606-1622Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The disruption of the Na+:K+:2Cl−cotransporter gene in humans (2.Simon D.B. Karet F.E. Di Hamdan J.M. Pietro A. Sanjad S.A. Lifton R.P. Nat. Genet. 1996; 13: 183-188Crossref PubMed Scopus (802) Google Scholar) and mice (3.Takahashi N. Chernavvsky D.R. Gomez R.A. Igarashi P. Gitelman H.J. Smithies O. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5434-5439Crossref PubMed Scopus (215) Google Scholar) 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+:K+:2Cl− cotransporter protein in the TALH is the main pharmacological target of loop diuretics (4.Hebert S.C. Windhager E.E. Handbook of Physiology: Renal Physiology. Oxford University Press, New York1992: 875-925Google Scholar), which are used extensively in the treatment of edematous states. thick ascending limb of Henle's loop cortical TALH medullary TALH bumetanide-sensitive cotransporter 1 (also known as NKCC2) bumetanide-sensitive Na+-K+-2Cl− cotransporter 2 (also known as NKCC1) mouse BSC1 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid R(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1-H-indenyl-5-yl)-oxy]acetic acid green fluorescent protein enhanced GFP tracer rubidium The primary structure of the kidney-specific, bumetanide-sensitive Na+:K+:2Cl− cotransporter (BSC1 or NKCC2) has been elucidated by cloning cDNA from rat (5.Gamba G. Miyanoshita A. Lombardi M. Lytton J. Lee W.S. Hediger M.A. Hebert S.C. J. Biol. Chem. 1994; 269: 17713-17722Abstract Full Text PDF PubMed Google Scholar), rabbit (6.Payne J A Forbush III B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4544-4548Crossref PubMed Scopus (260) Google Scholar), mouse (7.Igarashi P Vanden Heuver G B Payne J A Forbush III B. Am. J. Physiol. Renal Fluid Electrolyte Physiol. 1995; 269: F406-F418Crossref Google Scholar), and human kidney (2.Simon D.B. Karet F.E. Di Hamdan J.M. Pietro A. Sanjad S.A. Lifton R.P. Nat. Genet. 1996; 13: 183-188Crossref PubMed Scopus (802) Google Scholar). BSC1 belongs to the superfamily of electroneutral cation-coupled chloride cotransporters for which eight genes have been identified (8.Gamba G. Curr. Opin. Nephrol. Hypertens. 2000; 9: 535-540Crossref PubMed Scopus (18) Google Scholar). Two of these genes encode for Na+:K+:2Cl− 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 ∼60%, 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.Payne J A Forbush III B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4544-4548Crossref PubMed Scopus (260) Google Scholar, 7.Igarashi P Vanden Heuver G B Payne J A Forbush III B. Am. J. Physiol. Renal Fluid Electrolyte Physiol. 1995; 269: F406-F418Crossref Google Scholar). 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.Mount D.B. Baekgard A. Hall A.E. Plata C. Xu J. Beier D.R. Gamba G. Hebert S.C. Am. J. Physiol. Renal Physiol. 1999; 276: F347-F358Crossref PubMed Google Scholar). 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.Mount D.B. Baekgard A. Hall A.E. Plata C. Xu J. Beier D.R. Gamba G. Hebert S.C. Am. J. Physiol. Renal Physiol. 1999; 276: F347-F358Crossref PubMed Google Scholar, 10.Gamba G. Am. J. Physiol. Renal Physiol. 2001; 281: F781-F794Crossref PubMed Google Scholar). The splicing at the COOH-terminal domain in mouse BSC1 has remarkable effects on the cotransporter properties. Whereas the three longer isoforms (A, B, and F) function as bumetanide-sensitive Na+:K+:2Cl− cotransporters which are partially inhibited by hypotonicity (5.Gamba G. Miyanoshita A. Lombardi M. Lytton J. Lee W.S. Hediger M.A. Hebert S.C. J. Biol. Chem. 1994; 269: 17713-17722Abstract Full Text PDF PubMed Google Scholar, 11.Plata C. Mount D.B. Rubio V. Hebert S.C. Gamba G. Am. J. Physiol. Renal Physiol. 1999; 276: F359-F366Crossref PubMed Google Scholar), the shorter isoform operates as a K+-independent, but nevertheless bumetanide-sensitive Na+:Cl− cotransporter that is activated by hypotonicity (12.Plata C. Meade P. Hall A.E. Welch R.C. Vazquez N. Hebert S.C. Gamba G. Am. J. Physiol. Renal Physiol. 2001; 280: F574-F582Crossref PubMed Google Scholar). Both transporters are equally sensitive to loop diuretics. In addition, the shorter isoform is sensitive to cAMP and exerts a dominant-negative effect upon the Na+:K+:2Cl− cotransporter which can be abrogated by cAMP (11.Plata C. Mount D.B. Rubio V. Hebert S.C. Gamba G. Am. J. Physiol. Renal Physiol. 1999; 276: F359-F366Crossref PubMed Google Scholar). Thus, splicing of the COOH-terminal domain changes the type and stoichiometry of the cotransported ions, the response to cell swelling, and provides a potential regulatory mechanism of the Na+:K+:2Cl−cotransporter activity. 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.Burg M.B. Kidney Int. 1982; 22: 454-464Abstract Full Text PDF PubMed Scopus (105) Google Scholar) and medullary TALH (mTALH) segments by Rocha and Kokko (14.Rocha A.S. Kokko J.P. J. Clin. Invest. 1973; 52: 612-623Crossref PubMed Scopus (268) Google Scholar) indicated that mTALH transports NaCl more rapidly than the cTALH but with greater diluting power in the cTALH (15.Reeves W.B. Molony D.A. Andreoli T.E. Am. J. Physiol. 1988; 255: F1145-F1154PubMed Google Scholar), suggesting heterogeneity of the transport properties along the TALH. Supporting this possibility, the apparent affinity for Cl− observed by Greger (16.Greger R. Scand. Audiol. Suppl. 1981; 14: 1-15PubMed Google Scholar), Hus-Citharel and Morel (17.Hus-Citharel A. Morel F. Pflügers Arch. 1986; 407: 421-427Crossref PubMed Scopus (24) Google Scholar), and Eveloff et al. (18.Eveloff J. Bayerdorffer E. Silva P. Kinne R. Pflügers Arch. 1981; 389: 263-270Crossref PubMed Scopus (49) Google Scholar), when cTALH was used as a source of the plasma membrane vesicles, was different from the apparent affinity obtained by Koenig et al. (19.Koenig B. Ricapito S. Kinne R. Pflügers Arch. 1983; 399: 173-179Crossref PubMed Scopus (83) Google Scholar) and Burnham et al. (20.Burnham C. Karlish S.J. Jorgensen P.L. Biochim. Biophys. Acta. 1985; 821: 461-469Crossref PubMed Scopus (32) Google Scholar) 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.Payne J A Forbush III B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4544-4548Crossref PubMed Scopus (260) Google Scholar, 7.Igarashi P Vanden Heuver G B Payne J A Forbush III B. Am. J. Physiol. Renal Fluid Electrolyte Physiol. 1995; 269: F406-F418Crossref Google Scholar, 21.Yang T. Huang Y.G. Singh I. Schnermann J. Briggs J.P. Am. J. Physiol. 1996; 271: F931-F939PubMed Google Scholar). 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+:K+:2Cl− 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+:K+:2Cl− cotransporter using the Xenopus laevis oocytes as an heterologous expression system. Our data revealed significant differences in the affinity for Na+, K+, and Cl− among isoforms as well as in the sensitivity to bumetanide and response to hypotonicity. 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 CaCl2, 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.Dumont J.N. J. Morphol. 1970; 136: 153-180Crossref Scopus (1428) Google Scholar) were injected with 50 nl of water or cRNA at a concentration of 0.5 μg/μl (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−-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.Gamba G. Saltzberg S.N. Lombardi M. Miyanoshita A. Lytton J. Hediger M.A. Brenner B.M. Hebert S.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2749-2753Crossref PubMed Scopus (345) Google Scholar). The cloning and preparation of mouse mBSC1 cDNA used in the study have been reported previously (9.Mount D.B. Baekgard A. Hall A.E. Plata C. Xu J. Beier D.R. Gamba G. Hebert S.C. Am. J. Physiol. Renal Physiol. 1999; 276: F347-F358Crossref PubMed Google Scholar). In brief, mBSC1-F and mBSC1-A isoforms were cloned by homology from a mouse outer medulla cDNA library, using the flounder thiazide-sensitive Na+:Cl− cotransporter cDNA as a probe (5.Gamba G. Miyanoshita A. Lombardi M. Lytton J. Lee W.S. Hediger M.A. Hebert S.C. J. Biol. Chem. 1994; 269: 17713-17722Abstract Full Text PDF PubMed Google Scholar, 9.Mount D.B. Baekgard A. Hall A.E. Plata C. Xu J. Beier D.R. Gamba G. Hebert S.C. Am. J. Physiol. Renal Physiol. 1999; 276: F347-F358Crossref PubMed Google Scholar). The short B cassette cDNA was lengthened by PCR and ligated into the BsmI and NsiI sites of the mBSC1-F isoform (9.Mount D.B. Baekgard A. Hall A.E. Plata C. Xu J. Beier D.R. Gamba G. Hebert S.C. Am. J. Physiol. Renal Physiol. 1999; 276: F347-F358Crossref PubMed Google Scholar). 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 3′-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 −80 °C until used. The function of the Na+:K+:2Cl− cotransporter was assessed by measuring tracer 86Rb+ uptake (PerkinElmer Life Sciences) in groups of at least 15 oocytes following this general protocol: a 30-min incubation in isotonic K+- and Cl−-free medium (in mm: 96 sodium gluconate, 6.0 calcium gluconate, 1.0 magnesium gluconate, 5 HEPES/Tris, pH 7.4) with 1 mm ouabain followed by a 60-min uptake period in the presence of Na+, K+, and Cl−. For most experiments the isotonic medium contained (in mm): 96 NaCl, 10 KCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES, pH 7.4, supplemented with 1 mmouabain and 2.0 μCi of 86Rb+. Because X. laevis oocytes express an endogenous Na+:K+:2Cl− cotransporter (5.Gamba G. Miyanoshita A. Lombardi M. Lytton J. Lee W.S. Hediger M.A. Hebert S.C. J. Biol. Chem. 1994; 269: 17713-17722Abstract Full Text PDF PubMed Google Scholar) every experiment included the appropriate groups of water-injected oocytes. To analyze the ion transport kinetics of the Na+:K+:2Cl− cotransporter isoforms, experiments were performed varying the concentrations of Na+, K+ and Cl−. For Na+ kinetics, the extracellular K+ and Cl− concentrations were fixed at 10 and 90 mm, respectively. For K+ kinetics, Na+ and Cl− were fixed at 90 mm, and for Cl− kinetics the Na+ and K+concentrations were fixed at 90 and 10 mm, respectively. To maintain osmolarity and ionic strength, N-methyl-d-glucamine was used as an Na+ and K+ substitute, and gluconate was used as a Cl− substitute. The transport kinetics for a single ion (Na+, K+, or Cl−) 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 86Rb+ uptake because of the injected mBSC1 isoform. Kinetic analysis was performed by estimating the EC50 values for each ion. The EC50 values were calculated from log[ion concentration] versus V/Vmax 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 10−9 to 10−4m. 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 ∼ 160 mosmol/kg. To prepare the solutions with 210 and 260 mosmol/kg we added 45 and 90 mmsucrose, 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 β-scintillation counting. 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.Mount D.B. Baekgard A. Hall A.E. Plata C. Xu J. Beier D.R. Gamba G. Hebert S.C. Am. J. Physiol. Renal Physiol. 1999; 276: F347-F358Crossref PubMed Google Scholar). 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 ×10, 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. 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 ± S.E. We and others (5.Gamba G. Miyanoshita A. Lombardi M. Lytton J. Lee W.S. Hediger M.A. Hebert S.C. J. Biol. Chem. 1994; 269: 17713-17722Abstract Full Text PDF PubMed Google Scholar, 24.Burnham C.E. Kidd J. Palfrey H.C. Am. J. Physiol. Renal Physiol. 1990; 259: F383-F388Crossref PubMed Google Scholar, 25.Shetlar R.E. Scholermann B. Morrison A.I. Kinne R.K.H. Biochim. Biophys. Acta. 1990; 1023: 184-190Crossref PubMed Scopus (15) Google Scholar, 26.Plata C. Rubio V. Gamba G. Arch. Med. Res. 2000; 31: 21-27Crossref PubMed Scopus (7) Google Scholar) have shown previously that Xenopus oocytes exhibit an endogenous expression of the bumetanide-sensitive Na+:K+:2Cl− cotransporter. As shown in Fig. 1,86Rb+ uptake in H2O-injected oocytes was 2,113 ± 346 pmol·oocyte−1·h−1 in control conditions and 417 ± 202 pmol·oocyte−1·h−1 in the presence of a 10−4m concentration of the loop diuretic bumetanide. Background 86Rb+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 86Rb+ uptake induced only by each mBSC1 isoform, in all experiments performed for this study,86Rb+ 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, 86Rb+ uptake in mBSC1-A-injected oocytes was 19,395 ± 1,997 pmol·oocyte−1·h−1, whereas in mBSC1-B oocytes it was 13,229 ± 1,640 pmol·oocyte−1·h−1, and in mBSC1-F it was 12,088 ± 1,561 pmol·oocyte−1·h−1. Thus 86Rb+ uptake in the mBSC1-A isoform is significantly higher than in mBSC1-B and mBSC1-F isoforms (p < 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 5′- and 3′-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+:K+:2Cl− 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. 2Eshows 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 ± 4,165; n = 48) than in those injected with mBSC1-A (48,888 ± 8,042; n = 50) or mBSC1-B (43,995 ± 8,495; n = 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. 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+transport kinetics of each isoform, and panels B, C, and D depict the Hill coefficient plots for Na+ in mBSC1-B, mBSC1-A, and mBSC1-F, respectively. The Na+ dependence of 86Rb+ uptake was assessed with fixed concentrations of K+ and Cl− at 10 and 96 mm, respectively, with changing concentrations of Na+ from 0 to 80 mm.86Rb+ uptake increased as the Na+concentration was increased until a plateau phase was reached, compatible with Michaelis-Menten behavior. Table I shows the EC50 and Hill coefficient values. The EC50 values for Na+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+transport kinetics of each isoform, and panels B, C, and D depict the Hill coefficient plots for K+ in mBSC1-B, mBSC1-A, and mBSC1-F, respectively. The experiments were performed with fixed concentrations of Na+ and Cl− at 96 mm, with increased concentrations of K+ from 0 to 10 mm. The86Rb+ uptake increased as the K+concentration increased in the extracellular medium until a plateau phase was reached. EC50 and Hill coefficients are shown in Table I. As with Na+ transport kinetics, the EC50 values observed in mBSC1-A and mBSC1-B were similar, whereas the EC50 for K+ in mBSC1-F isoform was higher. Fig. 5A depicts the Cl− transport kinetics for each mBSC1 isoform, and panels B, C, and D show the Hill plots for Cl−. These experiments were carried out with Na+ and K+ concentrations fixed at 96 and 10 mm, respectively, with increased Cl−concentrations from 0 to 96 mm.86Rb+ uptake increased as a function of the Cl− concentration. The plateau phase was reached in mBSC1-A and mBSC1-B, but not in mBSC1-F. As shown in Table I, the EC50 value for Cl− was higher in mBSC1-F than in mBSC1-A or mBSC1-B. Hill coefficients for Na+ and K+ in the three isoforms were close to unity, whereas Hill coefficients for Cl− were above unity, consistent with the 1Na+, 1K+, and 2Cl− stoichiometry. As Figure 3, Figure 4, Figure 5 show, in general mBSC1-A and mBSC1-B exhibit very similar kinetic properties for the three cotransported ions, suggesting that affinity for each ion is similar between these two isoforms. In contrast, the EC50 values for Na+, K+, and Cl− in mBSC1-F-injected oocytes were higher, suggesting that this is the isoform with the lowest affinity for the cotransported ions.Table IEC50 values and Hill coefficient for Na+,K+, and Cl− transport in mBSC1 isoformsSodiumPotassiumChlorideEC50HillEC50HillEC50HillmBSC1-B3.0 ± 0.61.09 ± 0.10.76 ± 0.071.00 ± 0.111.6 ± 0.71.53 ± 0.06mBSC1-A5.0 ± 3.91.16 ± 0.10.96 ± 0.160.83 ± 0.0922.2 ± 4.81.93 ± 0.31mBSC1-F20.6 ± 7.20.78 ± 0.11.54 ± 0.160.95 ± 0.0529.2 ± 2.12.85 ± 0.25 Open table in a new tab Figure 4Kinetic transport analysis for K+in mBSC1 isoforms. Panel A, K+-dependent 86Rb+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+ concentrations of 0.1, 0.25, 0.4, 0.6, 1.0, 2, 5, and 10 mm. For the K+ kinetics analysis the Na+ and Cl− concentration was fixed at 96 mm. Lines were fit using the Michaelis-Menten equation. Each point represents the mean ± S.E. of 15 oocytes. Panels B, C, and D show the Hill plots for K+ in mBSC1-B, mBSC1-A, and mBSC1-F, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Kinetic transport analysis for Cl− in mBSC1 isoforms. Panel A, Cl−-dependent86Rb+ uptake in oocytes injected with mBSC1-A (circles), mBSC1-B (boxes), and mBSC1-F (triangles) cRNA. Uptake was assessed in the presence of increased concentrations of extracellular Cl− of 2.5, 5, 12, 20, 40, 60, 80, and 100 mm, with the concentration of Na+ and K+ fixed at 96 and 10 mm, respectively. Lines were fit using the Michaelis-Menten equation. Each point represents the mean ± S.E. of 15 oocytes. Panels B, C, and D show the Hill plots for Cl− in mBSC1-B, mBSC1-A, and mBSC1-F, respectively.Vie" @default.
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• W1966841727 title "Functional Properties of the Apical Na+-K+-2Cl− Cotransporter Isoforms" @default.
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