Comparison of Na-K-Cl Cotransporters
1998; Elsevier BV; Volume: 273; Issue: 18 Linguagem: Inglês
10.1074/jbc.273.18.11295
ISSN1083-351X
AutoresPaul Isenring, Steven C. Jacoby, John A. Payne, Bliss Forbush,
Tópico(s)Ion Transport and Channel Regulation
ResumoThe Na-K-Cl cotransporter (NKCC) mediates the coupled movement of ions into most animal cells, playing important roles in maintenance of cell volume and in epithelial Cl transport. Two forms of NKCC have been described: NKCC1, the "housekeeping" isoform that is also responsible for Cl accumulation in secretory epithelial cells, and NKCC2, which mediates apical Na+K+Cl entry into renal epithelial cells. Here we examine the kinetic properties of NKCC1, NKCC2, and the endogenous HEK-293 cell cotransporter. Stable expression of rabbit NKCC2A was obtained in HEK-293 cells utilizing a chimera (h1r2A0.7) in which the 5′-untranslated region and cDNA encoding 104 amino acids of the N terminus are replaced by the corresponding sequence of NKCC1. h1r2A0.7 exhibits Na and Cl affinities near those of NKCC1, but it has a 4-fold lower Rb affinity, and a 3-fold higher affinity for the inhibitor bumetanide. The activity of h1r2A0.7 is increased on incubation in low [Cl] media as is NKCC1, but the resting level of activity is higher in h1r2A0.7 and activation is more rapid. h1r2A0.7 exhibits an appropriate volume response, unlike NKCC1 for which concomitant changes in [Cl]i appear to be the overriding factor. These results support a model in which apical NKCC2 activity is matched to basolateral Cl exit through changes in [Cl]i. Reverse transcriptase-polymerase chain reaction of HEK-293 cell mRNA is positive with NKCC1 primers and negative with NKCC2 primers. Surprisingly, we found that the behavior of the endogenous HEK cell Na-K-Cl cotransporter is unlike either of the two forms which have been described: compared with NKCC1, HEK cell cotransporter has a 2.5-fold lower Na affinity, an 8-fold lower Rb affinity, and a 4-fold higher bumetanide affinity. These results suggest the presence of a novel isoform of NKCC in HEK-293 cells. The Na-K-Cl cotransporter (NKCC) mediates the coupled movement of ions into most animal cells, playing important roles in maintenance of cell volume and in epithelial Cl transport. Two forms of NKCC have been described: NKCC1, the "housekeeping" isoform that is also responsible for Cl accumulation in secretory epithelial cells, and NKCC2, which mediates apical Na+K+Cl entry into renal epithelial cells. Here we examine the kinetic properties of NKCC1, NKCC2, and the endogenous HEK-293 cell cotransporter. Stable expression of rabbit NKCC2A was obtained in HEK-293 cells utilizing a chimera (h1r2A0.7) in which the 5′-untranslated region and cDNA encoding 104 amino acids of the N terminus are replaced by the corresponding sequence of NKCC1. h1r2A0.7 exhibits Na and Cl affinities near those of NKCC1, but it has a 4-fold lower Rb affinity, and a 3-fold higher affinity for the inhibitor bumetanide. The activity of h1r2A0.7 is increased on incubation in low [Cl] media as is NKCC1, but the resting level of activity is higher in h1r2A0.7 and activation is more rapid. h1r2A0.7 exhibits an appropriate volume response, unlike NKCC1 for which concomitant changes in [Cl]i appear to be the overriding factor. These results support a model in which apical NKCC2 activity is matched to basolateral Cl exit through changes in [Cl]i. Reverse transcriptase-polymerase chain reaction of HEK-293 cell mRNA is positive with NKCC1 primers and negative with NKCC2 primers. Surprisingly, we found that the behavior of the endogenous HEK cell Na-K-Cl cotransporter is unlike either of the two forms which have been described: compared with NKCC1, HEK cell cotransporter has a 2.5-fold lower Na affinity, an 8-fold lower Rb affinity, and a 4-fold higher bumetanide affinity. These results suggest the presence of a novel isoform of NKCC in HEK-293 cells. The Na-K-Cl cotransporter (NKCC or BSC) 1The abbreviations used are: NKCC, Na-K-Cl cotransporter; CCC, cation-chloride cotransporter; KCC, K-Cl cotransporter; TAL, thick ascending limb; 5′-UTR, 5′-untranslated region; RT-PCR, reverse transcriptase-polymerase chain reaction; bp, base pair(s); m, h, s, and rNKCC, mouse, human, shark, and rabbit NKCC, respectively. mediates the coupled movement of Na, K, and Cl ions across the plasma membrane of animal cells. The transporter plays an important role in electrolyte movement across polarized epithelia and is also thought to be involved in regulation of intracellular volume and intracellular [Cl] (1Haas M. Annu. Rev. Physiol. 1989; 51: 443-457Crossref PubMed Scopus (193) Google Scholar, 2Lytle C. Forbush III, B. Am. J. Physiol. 1996; 39: C437-C448Crossref Google Scholar). NKCC is a member of the Na-coupled group of cation-chloride cotransporters (CCCs) (1Haas M. Annu. Rev. Physiol. 1989; 51: 443-457Crossref PubMed Scopus (193) Google Scholar, 3Gillen C.M. Brill S. Payne J.A. Forbush III, B. J. Biol. Chem. 1996; 271: 16237-16244Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar), a family which also includes K-Cl cotransporters (KCC) (3Gillen C.M. Brill S. Payne J.A. Forbush III, B. J. Biol. Chem. 1996; 271: 16237-16244Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 4Payne J.A. Stevenson T.J. Donaldson L.F. J. Biol. Chem. 1996; 271: 16245-16252Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar). Three Na-coupled cation-chloride cotransporters have been described to date. 1) The "secretory" (or "housekeeping" or "basolateral") Na-K-Cl cotransporter, NKCC1 (or BSC2), is widely distributed in mammalian tissues (5Delpire E. Rauchman M.I. Beier D.R. Hebert S.C. Gullans S.R. J. Biol. Chem. 1994; 269: 25677-25683Abstract Full Text PDF PubMed Google Scholar, 6Payne J.A. Xu J.C. Haas M. Lytle C.Y. Ward D. Forbush III, B. J. Biol. Chem. 1995; 270: 17977-17985Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar) and is especially prominent in the basolateral membranes of secretory epithelial cells; within the kidney, NKCC1 is found in epithelial cells in the collecting duct and in the glomerulus (7Ginns S.M. Knepper M.A. Ecelbarger C.A. Terris J. He X. Coleman R.A. Wade J.B. J. Am. Soc. Nephrol. 1996; 7: 2533-2542PubMed Google Scholar, 8Kaplan M.R. Plotkin M.D. Brown D. Hebert S.C. Delpire E. J. Clin. Invest. 1996; 98: 723-730Crossref PubMed Scopus (124) Google Scholar). 2) The "renal" or "apical" Na-K-Cl cotransporter, NKCC2 (or BSC1) (9Payne J.A. Forbush III, B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4544-4548Crossref PubMed Scopus (261) Google Scholar, 10Gamba 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), is found only in the apical membrane of epithelial cells in the thick ascending limb of the loop of Henle (TAL) (11Igarashi P. Vanden Heuvel G.B. Payne J.A. Forbush III, B. Am. J. Physiol. 1995; 269: F405-F418PubMed Google Scholar, 12Kaplan M.R. Plotkin M.D. Lee W.S. Xu Z.C. Lytton J. Hebert S.C. Kidney Int. 1996; 49: 40-47Abstract Full Text PDF PubMed Scopus (160) Google Scholar, 13Ecelbarger C.A. Terris J. Hoyer J.R. Nielsen S. Wade J.B. Knepper M.A. Am. J. Physiol. 1996; 271: F619-F628Crossref PubMed Google Scholar, 14Obermuller N. Kunchaparty S. Ellison D.H. Bachmann S. J. Clin. Invest. 1996; 98: 635-640Crossref PubMed Scopus (99) Google Scholar, 15Yang T. Huang Y.G. Singh I. Schnermann J. Briggs J.P. Am. J. Physiol. 1996; 271: F931-F939PubMed Google Scholar). Three splice variants of NKCC2 (A, B, and F), differing in the sequence of the second predicted transmembrane domain, are differentially distributed along the nephron (9Payne J.A. Forbush III, B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4544-4548Crossref PubMed Scopus (261) Google Scholar, 11Igarashi P. Vanden Heuvel G.B. Payne J.A. Forbush III, B. Am. J. Physiol. 1995; 269: F405-F418PubMed Google Scholar). 3) The Na-Cl cotransporter, NCC (or TSC) (16Gamba 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 (349) Google Scholar), is restricted to the apical membrane of the distal tubule in the mammalian kidney. The activity of the Na-K-Cl cotransporter is increased in most cells in response to cell shrinkage, leading to a regulatory increase in cell volume (1Haas M. Annu. Rev. Physiol. 1989; 51: 443-457Crossref PubMed Scopus (193) Google Scholar). Additionally, in secretory epithelia, cotransporter activity is strongly regulated as part of the process controlling fluid secretion: it appears that a decrease in intracellular [Cl] is the message which triggers an increase in cotransport activity and thereby achieves apical-basolateral communication (17Lytle C. Forbush III, B. J. Biol. Chem. 1992; 267: 25438-25443Abstract Full Text PDF PubMed Google Scholar, 18Haas M. McBrayer D.G. Am. J. Physiol. 1994; 266: C1440-C1452Crossref PubMed Google Scholar, 19Robertson M.A. Foskett J.K. Am. J. Physiol. 1994; 267: C146-C156Crossref PubMed Google Scholar, 20Lytle C. Forbush III, B. Am. J. Physiol. 1996; 270: C437-C448Crossref PubMed Google Scholar). We have shown that for NKCC1, modulation of transport in response to both volume change and [Cl]i change involves direct phosphorylation of the NKCC1 protein (17Lytle C. Forbush III, B. J. Biol. Chem. 1992; 267: 25438-25443Abstract Full Text PDF PubMed Google Scholar, 20Lytle C. Forbush III, B. Am. J. Physiol. 1996; 270: C437-C448Crossref PubMed Google Scholar). We have recently used chimeras of human and shark NKCC1 to identify regions that are responsible for mediating the binding characteristics of the transporters, taking advantage of 5-fold species differences in kinetic constants for ion translocation and bumetanide inhibition (21Isenring P. Forbush III, B. J. Biol. Chem. 1997; 272: 24556-24562Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). When the N and the C termini were interchanged between species, we found no significant change in kinetic parameters, indicating that it is the large central transmembrane domain of the NKCC protein that encodes the differences in ion and bumetanide binding. The function of NKCC has been studied by expression of transporter cDNAs in mammalian cell lines (22Xu J. Lytle C. Zhu T.T. Payne J.A. Benz Jr., E. Forbush III, B. Proc. Natl. Acad. Sci. 1994; 91: 2201-2205Crossref PubMed Scopus (376) Google Scholar) and in Xenopusoocytes (10Gamba 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). Mammalian expression systems offer considerable advantages in reproducibility and in the ability to perform assays under a large number of conditions. We have determined the characteristics of NKCC1-mediated transport using stable expression in HEK-293 cells (6Payne J.A. Xu J.C. Haas M. Lytle C.Y. Ward D. Forbush III, B. J. Biol. Chem. 1995; 270: 17977-17985Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 23Forbush III, B. Haas M. Lytle C. Am. J. Physiol. 1992; 262: C1000-C1008Crossref PubMed Google Scholar) but unfortunately have been unable to obtain functional expression of NKCC2 using the same methods (9Payne J.A. Forbush III, B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4544-4548Crossref PubMed Scopus (261) Google Scholar). Similarly, it has been difficult to express NKCC1 in oocytes (5Delpire E. Rauchman M.I. Beier D.R. Hebert S.C. Gullans S.R. J. Biol. Chem. 1994; 269: 25677-25683Abstract Full Text PDF PubMed Google Scholar). In this project we have been able to characterize ion transport mediated by NKCC2 utilizing a chimera (h1r2A0.7) in which 104 amino acids of the N terminus are replaced by corresponding residues of NKCC1. Apparently, translation or processing efficiency is higher with the NKCC1 5′-UTR and N terminus. Most of this region is very poorly conserved from one species to another and from one isoform to another, both for NKCC and KCC (3Gillen C.M. Brill S. Payne J.A. Forbush III, B. J. Biol. Chem. 1996; 271: 16237-16244Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 24Payne J.A. Forbush III, B. Curr. Opin. Cell Biol. 1995; 7: 493-503Crossref PubMed Scopus (108) Google Scholar). Since our previous experiments demonstrate that neither the N nor the C terminus contributes to the differences in ion affinities between sNKCC1 and hNKCC1 (21Isenring P. Forbush III, B. J. Biol. Chem. 1997; 272: 24556-24562Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), we do not anticipate that the N-terminal change in h1r2A0.7 significantly alters the function of NKCC2. The HEK-293 cell line used for expression of NKCC in this and previous studies is derived from human embryonic kidney, immortalized by adenovirus transformation (25Graham F.L. Smiley J. Russell W.C. Nairn R. J. Gen. Virol. 1977; 36: 59-74Crossref PubMed Scopus (3667) Google Scholar). HEK cells have a rather low level of endogenous ion fluxes, including Na-K-Cl cotransport. The de-differentiated line does not exhibit epithelial characteristics, and it is therefore not possible to predict which isoform of Na-K-Cl cotransporter might be present. In this study, we compare the kinetic and regulatory behavior of NKCC1, h1r2A0.7, and the endogenous HEK cell cotransporter. We find that NKCC1 and NKCC2 (as h1r2A0.7) are different from one another in ion and bumetanide affinities as well as in the relative sensitivities to cell volume and [Cl]. Surprisingly, we find also that the endogenous Na-K-Cl cotransporter in HEK cells exhibits unique functional features, its behavior being different from that of both NKCC1 and NKCC2. Part of this work has been previously reported in abstract form (26Payne J.A. Gillen C. Forbush III, B. J. Am. Soc. Nephrol. 1995; 6: 348Google Scholar). h1r2A0.7 is composed of the entire coding region of the renal Na-K-Cl cotransporter (rNKCC2A), except that two-thirds of the N terminus is replaced by the corresponding region in hNKCC1 (Fig. 1). A common NcoI restriction site in hNKCC1 and rNKCC2A was used to create the chimeric junction 0.7 (Thr217/Met218 in hNKCC1 and Thr104/Met105 in rNKCC2A). This site occurs in a conserved region in the N terminus, 75 amino acids before the first putative transmembrane domain. The cDNA was prepared by simultaneous ligation of four fragments into the pJB20 expression vector (27Beck P.J. Orlean P. Albright C. Robbins P.W. Gething M.J. Sambrook J.F. Mol. Cell. Biol. 1990; 10: 4612-4622Crossref PubMed Scopus (58) Google Scholar) (6200-bp length) at EcoRI-KpnI sites. The fragments were: EcoRI-NcoI from hNKCC1 (900-bp length); and NcoI-SphI (250-bp length),SphI-XbaI (1440-bp length), andXbaI-KpnI (1650-bp length) from rNKCC2A. The final construct was analyzed by automated sequencing and restriction analysis. Control HEK cells, mock-transfected cells, and lines stably expressing sNKCC1 and hNKCC1 were the same as in Ref. 21Isenring P. Forbush III, B. J. Biol. Chem. 1997; 272: 24556-24562Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar. h1r2A0.7 cDNA was transfected into HEK cells by calcium phosphate precipitation, and stable lines were isolated by G-418 resistance, as described previously (6Payne J.A. Xu J.C. Haas M. Lytle C.Y. Ward D. Forbush III, B. J. Biol. Chem. 1995; 270: 17977-17985Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 21Isenring P. Forbush III, B. J. Biol. Chem. 1997; 272: 24556-24562Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). T84 cells, obtained from J. Madara, were as in Ref. 6Payne J.A. Xu J.C. Haas M. Lytle C.Y. Ward D. Forbush III, B. J. Biol. Chem. 1995; 270: 17977-17985Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar. NIH-3T3 cells and the E12a mutant (28Sussman I. O'Brien T.G. J. Cell Physiol. 1985; 124: 153-159Crossref PubMed Scopus (14) Google Scholar) were from T. G. O'Brien. All lines were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin, streptomycin, and G-418 (for transfected cells), as described previously (21Isenring P. Forbush III, B. J. Biol. Chem. 1997; 272: 24556-24562Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). We have studied two lines of h1r2A0.7 from separate transfections; the results for the two lines were indistinguishable from one another, and we have combined the data as reported here. Similarly, several experiments with hNKCC1 were performed with a different line reported in Ref. 6Payne J.A. Xu J.C. Haas M. Lytle C.Y. Ward D. Forbush III, B. J. Biol. Chem. 1995; 270: 17977-17985Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, with indistinguishable results. All experiments with sNKCC1 were carried out with the cell line described in Ref. 21Isenring P. Forbush III, B. J. Biol. Chem. 1997; 272: 24556-24562Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar. This line appears to be functionally the same as a line described in Ref. 22Xu J. Lytle C. Zhu T.T. Payne J.A. Benz Jr., E. Forbush III, B. Proc. Natl. Acad. Sci. 1994; 91: 2201-2205Crossref PubMed Scopus (376) Google Scholar except that the cotransport flux is somewhat higher. 86Rb influx in HEK cells was determined as described previously (6Payne J.A. Xu J.C. Haas M. Lytle C.Y. Ward D. Forbush III, B. J. Biol. Chem. 1995; 270: 17977-17985Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 21Isenring P. Forbush III, B. J. Biol. Chem. 1997; 272: 24556-24562Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Briefly, cells were grown to confluence (6–8 days at 37 °C) in polylysine-coated 96-well plates. In most experiments, cells were preincubated in hypotonic low Cl medium (regular flux medium diluted 1:2 with water and with gluconate replacing all but 2.5 mm Cl) to activate the cotransporter (6Payne J.A. Xu J.C. Haas M. Lytle C.Y. Ward D. Forbush III, B. J. Biol. Chem. 1995; 270: 17977-17985Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 22Xu J. Lytle C. Zhu T.T. Payne J.A. Benz Jr., E. Forbush III, B. Proc. Natl. Acad. Sci. 1994; 91: 2201-2205Crossref PubMed Scopus (376) Google Scholar). Fluxes were carried out for 1 min at room temperature (≃ 22 °C) in "regular flux medium" that contained 135 mm NaCl, 5 mm RbCl (2 μCi/ml86Rb), 1 mm CaCl2, 1 mmMgCl2, 1 mm Na2HPO4, 1 mm Na2SO4, 15 mmNa-HEPES, pH 7.4 and, when used for the flux assay but not for preincubations, 0.1 mm ouabain. Each concentration curve was carried out in a single row of the 96-well plate, and in each experiment there were 2–6 replicate rows. Counts of the 1-min 86Rb influx were normalized to the value at the highest ion concentration, or to the longest time point in an activation time course, or to the value of uninhibited flux in inhibition studies. In previous experiments, we determined that at confluence, the coefficient of standard variation of protein content in several wells is quite small, approximately 10% of the mean. This coefficient is similar to that calculated for absolute counts in several wells under the same conditions, and therefore, it has not been useful to routinely determine protein on a well-by-well basis. Rows with obvious rogue values were omitted from averages, which in all cases was less than one row in 10. Data are expressed as means ± S.E. among all rows in several experiments (on average 20–30 rows in 5–8 experiments). Similarly, K m andK i values were obtained on a per row basis by non-linear least squares curve fitting using the Simplex algorithm (program PLOT, B. Forbush). Where error bars are not visible, they are smaller than the symbols. We have not attempted to correct fluxes in transfected cells for a potential background contribution from HEK cell cotransporter because we have evidence that expression of exogenous cotransporter suppresses endogenous cotransporter. 2R. Behnke, P. Isenring, and B. Forbush, unpublished data. Poly(A)-selected RNA was isolated from T84 cells, 3T3 fibroblasts, E12a cells, and HEK cells. Confluent cells from 10-cm dishes were homogenized and digested for 1 h in 200 μg/ml proteinase K, 0.5% SDS, 100 mmNaCl, 20 mm Tris-Cl and 1 mm EDTA, at pH 8.0 and at 37 °C. After adjusting NaCl concentration to 400 mm, the cell lysates were incubated with oligo(dT)-cellulose for 4 h at room temperature. Poly(A) RNA was eluted in 1 mm EDTA, 0.05% SDS, pH 8.0, and concentrated by ethanol precipitation. Poly(A) RNA was primed with a gene-specific antisense oligonucleotide derived from a conserved region in the third transmembrane domain of either NKCC1 (gaatcacgactgtaatggctccaa; base pair 1422) or NKCC2 (ctccagagatgttggcaccagcaag); bp 1487). The primers were extended with the enzyme AMV in an appropriate reaction buffer with 0.4 μm dNTPs. For PCR, a sense oligonucleotide derived from another conserved sequence in the N terminus of NKCC1 (gcgcaccttcggccacaacaccatg; bp 794) or NKCC2 (ccgagttcggtgggtcaataggcttg; bp 1041) was added to the first cDNA strand, along with the DNA polymerases Taq and PWO. The NKCC1 primers correspond to regions that are identical between human and mouse NKCC1; NKCC2 primers correspond to a region identical in mNKCC2 and rNKCC2 and with 1 and 2 mismatches in hNKCC2. The NKCC1 target sequences are less than 20% identical to corresponding NKCC2 sequences, and NKCC2 target sequences are, respectively, less than 20 and 50% identical to corresponding NKCC1 regions. The region of interest (630 bp in NKCC1 and 440 bp in NKCC2) was amplified through 40 cycles of PCR with denaturation steps of 40 s at 95 °C, annealing 50 s at 50 °C, and extension 1 min at 70 °C. To test for amplification yields, 0.1 ng of plasmid DNA (either hNKCC1/Bluescript SK− or rNKCC2A/Bluescript SK−) was processed in the same manner as the poly(A) RNA, including an incubation period with reverse transcriptase. Samples of PCR reactions were analyzed on a 1.2% agarose gel stained with ethidium bromide (10 μg/ml). In previous efforts we have been unable to measure ion fluxes of rNKCC2 transfected in mammalian cell lines (COS cells, HEK, 3T3, Madin-Darby canine kidney (MDCK)), 3J. Payne and B. Forbush, unpublished data. apparently because of low levels of expression or poor cell surface delivery (9Payne J.A. Forbush III, B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4544-4548Crossref PubMed Scopus (261) Google Scholar). Also, truncation and modification of the 5′-UTR of rNKCC2 did not increase functional expression.3 We have successfully approached the problem using a chimera, h1r2A0.7, in which the 5′-UTR and cDNA encoding the first 104 amino acids of rNKCC2A were replaced with the corresponding region from hNKCC1. The N terminus of the cation-chloride cotransporters is very poorly conserved across isoforms and species (3Gillen C.M. Brill S. Payne J.A. Forbush III, B. J. Biol. Chem. 1996; 271: 16237-16244Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 24Payne J.A. Forbush III, B. Curr. Opin. Cell Biol. 1995; 7: 493-503Crossref PubMed Scopus (108) Google Scholar), and we have shown that it does not play a role in sNKCC1/hNKCC1 ion affinity differences (21Isenring P. Forbush III, B. J. Biol. Chem. 1997; 272: 24556-24562Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). When stimulated by preincubation in low [Cl] medium (see below), h1r2A0.7-transfected cell lines were found to transport 86Rb about 2.5-fold faster than control HEK cells (data not shown). 4We have been unable to obtain functional expression of B and F splice variants of NKCC2 using similar chimeric constructs (J. Payne and B. Forbush, unpublished data). This is a lower level of transport than obtained on transfection of sNKCC1 (4–6-fold above control) or hNKCC1 (7–9-fold above control), but as will be seen below, the properties of h1r2A0.7 and HEK-cell cotransporter are readily distinguished. To compare the transport behavior of NKCC isoforms, we measured the dependence of86Rb influx on Na, Rb, Cl, and bumetanide concentration for sNKCC1, hNKCC1, h1r2A0.7, as well as for HEK cells. The results are illustrated in Figs. 2 and 4, and theK m for Na, Rb, and Cl and the K i for bumetanide are summarized in Fig. 3. As has been consistently noted for the Na-K-Cl cotransporter, Na, Rb, and bumetanide dependences of 86Rb influx fit a model of ligand binding at a single site (Fig. 2), whereas the relation between [Cl] and 86Rb influx is sigmoidal, consistent with two binding and translocation sites for Cl (Fig. 4). The data generally agree well with values that have been previously reported by this laboratory in separate studies (6Payne J.A. Xu J.C. Haas M. Lytle C.Y. Ward D. Forbush III, B. J. Biol. Chem. 1995; 270: 17977-17985Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 21Isenring P. Forbush III, B. J. Biol. Chem. 1997; 272: 24556-24562Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar,22Xu J. Lytle C. Zhu T.T. Payne J.A. Benz Jr., E. Forbush III, B. Proc. Natl. Acad. Sci. 1994; 91: 2201-2205Crossref PubMed Scopus (376) Google Scholar). 5The values forK i (bumetanide) are rather different from one report to another; the values reported here are 2-fold higher than in Ref. 22Xu J. Lytle C. Zhu T.T. Payne J.A. Benz Jr., E. Forbush III, B. Proc. Natl. Acad. Sci. 1994; 91: 2201-2205Crossref PubMed Scopus (376) Google Scholar and Ref. 6Payne J.A. Xu J.C. Haas M. Lytle C.Y. Ward D. Forbush III, B. J. Biol. Chem. 1995; 270: 17977-17985Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar and 5Delpire E. Rauchman M.I. Beier D.R. Hebert S.C. Gullans S.R. J. Biol. Chem. 1994; 269: 25677-25683Abstract Full Text PDF PubMed Google Scholar-fold higher than in Ref. 21Isenring P. Forbush III, B. J. Biol. Chem. 1997; 272: 24556-24562Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar. Our procedures remain the same, and we speculate that the difference is due to differences in the lots of bumetanide (recently obtained from Sigma; in earlier work, also from P. Feit and from Hoechst); we have not yet tested this hypothesis. Despite these absolute differences, the relative differences from one isoform or species to another are similar within each set of determinations. We also note that the value ofK m (Cl) for sNKCC1 in Ref. 6Payne J.A. Xu J.C. Haas M. Lytle C.Y. Ward D. Forbush III, B. J. Biol. Chem. 1995; 270: 17977-17985Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar is 0.6 of that reported in Ref. 22Xu J. Lytle C. Zhu T.T. Payne J.A. Benz Jr., E. Forbush III, B. Proc. Natl. Acad. Sci. 1994; 91: 2201-2205Crossref PubMed Scopus (376) Google Scholar, Ref. 21Isenring P. Forbush III, B. J. Biol. Chem. 1997; 272: 24556-24562Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, and this work, and we presume that the value in Ref. 6Payne J.A. Xu J.C. Haas M. Lytle C.Y. Ward D. Forbush III, B. J. Biol. Chem. 1995; 270: 17977-17985Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar is inaccurate. Figure 4Bumetanide inhibition curves.After preincubation in low Cl (5 mm) hypotonic medium, cells were incubated for 15 min in reduced Cl (20 mm) medium at various bumetanide concentrations (6Payne J.A. Xu J.C. Haas M. Lytle C.Y. Ward D. Forbush III, B. J. Biol. Chem. 1995; 270: 17977-17985Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 22Xu J. Lytle C. Zhu T.T. Payne J.A. Benz Jr., E. Forbush III, B. Proc. Natl. Acad. Sci. 1994; 91: 2201-2205Crossref PubMed Scopus (376) Google Scholar) and then assayed for 86Rb influx in regular flux medium. The data (37–42 flux rows from six to seven experiments) are fit by a model of bumetanide inhibition at a single site.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3K m for Na, Rb, and Cl. Values were obtained from fits of the data in Fig. 2 and from similar data for sNKCC1 (18–29 rows from three to five experiments) and mock-transfected HEK cells (29–41 rows from five to eight experiments). The data for individual flux rows were analyzed by non-linear least squares fits, means ± S.E. of theK i and K m values are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The kinetic data demonstrate that NKCC2A (as h1r2A0.7) presents distinct ligand binding characteristics. The K m of h1r2A0.7 for Na is 40% lower than that of hNKCC1, and the K m for Rb is 4-fold higher compared with hNKCC1. In comparison to previous measurements ofK m values for ion transport in mouse kidney cortical TAL (29Greger R. Physiol. Rev. 1985; 65: 760-797Crossref PubMed Scopus (587) Google Scholar) and in a mouse medullary TAL cell line (30Kaji D.M. Biochim. Biophys. Acta. 1993; 1152: 289-299Crossref PubMed Scopus (15) Google Scholar),K m (Cl) is in the same range (34 compared with ≃50 and 67 mm, respectively),K m (Rb) is significantly greater (8 compared with 1–2 and 1.3 mm), andK m (Na) is similar to that reported for the TAL line (10 versus 7 mm) but greater than recorded in the cortical TAL (2–3 mm). While some of these differences may be attributable to methodology, it is also possible that they reflect differences in ion affinities of the three splice variants of NKCC2 that are differentially distributed along the TAL (9Payne J.A. Forbush III, B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4544-4548Crossref PubMed Scopus (261) Google Scholar,11Igarashi P. Vanden Heuvel G.B. Payne J.A. Forbush III, B. Am. J. Physiol. 1995; 269: F405-F418PubMed Google Scholar). In examining the kinetic behavior of endogenous HEK cell cotransport, we were surprised to find that it was significantly different both from NKCC1 (noted previously
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