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• W2087372011 abstract "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) 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. 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 (173) 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 (173) 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 (337) 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 (337) 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 (456) 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 (224) 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 (254) 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 (158) 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 (98) 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 (254) 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 (338) 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 (173) 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 (106) 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 (368) 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 (224) 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 (254) 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 (337) Google Scholar, 24Payne J.A. Forbush III, B. Curr. Opin. Cell Biol. 1995; 7: 493-503Crossref PubMed Scopus (107) 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 (106) 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 (3468) 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).DISCUSSIONThe results presented here provide a comparison of kinetic characteristics of hNKCC1, a rNKCC2A chimera, and the native HEK cell cotransporter. These kinetic parameters are summarized in TableI. As considered above, the characteristics of ion and bumetanide binding as well as the characteristics of regulation are different for each of the cotransporter forms.Table IKinetic parameters for NKCChNKCC1h1r2A0.7HEK-293sNKCC1K m (mm) Na14.57 ± 1.241-ap < 0.01 compared to all the other cell lines.9.79 ± 1.231-ap < 0.01 compared to all the other cell lines.34.69 ± 6.381-ap < 0.01 compared to all the other cell lines.101.3 ± 11.91-ap < 0.01 compared to all the other cell lines. Rb1.95 ± 0.091-ap < 0.01 compared to all the other cell lines.8.10 ± 0.971-ap < 0.01 compared to all the other cell lines.15.19 ± 2.9215.15 ± 1.59 Cl31.25 ± 0.7934.15 ± 0.7941.93 ± 2.011-ap < 0.01 compared to all the other cell lines.107.9 ± 8.21-ap < 0.01 compared to all the other cell lines.K i (bumetanide) (μm)0.28 ± 0.011-ap < 0.01 compared to all the other cell lines.0.08 ± 0.010.08 ± 0.011.04 ± 0.071-ap < 0.01 compared to all the other cell lines.K i (Hg) (μm)56.15 ± 2.991-ap < 0.01 compared to all the other cell lines.133.1 ± 23.791-ap < 0.01 compared to all the other cell lines.25.65 ± 1.571-ap < 0.01 compared to all the other cell lines.t½ in low Cli12 min2 min1-ap < 0.01 compared to all the other cell lines.6 min1-ap < 0.01 compared to all the other cell lines.12 minResting activity levels2 ± 3%22 ± 3%1-ap < 0.01 compared to all the other cell lines.14 ± 2%1-ap < 0.01 compared to all the other cell lines.4 ± 1%1-a p < 0.01 compared to all the other cell lines. Open table in a new tab This work provides the first detailed description of an isolated form of NKCC2. NKCC2A is found in the apical membrane of the thick ascending limb of the loop of Henle in the mammalian kidney (11Igarashi P. Vanden Heuvel G.B. Payne J.A. Forbush III, B. Am. J. Physiol. 1995; 269: F405-F418PubMed 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, 15Yang T. Huang Y.G. Singh I. Schnermann J. Briggs J.P. Am. J. Physiol. 1996; 271: F931-F939PubMed Google Scholar), specifically in segments found in the outer stripe of the outer medulla and in the inner cortex (11Igarashi P. Vanden Heuvel G.B. Payne J.A. Forbush III, B. Am. J. Physiol. 1995; 269: F405-F418PubMed Google Scholar). Our results demonstrate that NKCC2A is kinetically different from hNKCC1, particularly in having a lower Rb affinity (K m ≃ 8 mm). This is a surprisingly high value, and it would appear to ensure that the cotransporter is not saturated with luminal K under physiological conditions. Compared with NKCC1, NKCC2A was also found to have severalfold greater sensitivity to the loop diuretic drug bumetanide, a difference that may be advantageous from the standpoint of drug efficacy.We have used a chimeric construct in order to obtain sufficient expression to measure NKCC2-mediated flux. In addition to the 5′-UTR from NKCC1, the h1r2A0.7 chimera includes hNKCC1 residues replacing the first 104 residues of the rabbit NKCC2A sequence. Most of the N terminus is very poorly conserved within the CCC family, and where we have tested, we have seen no effect of N-terminal modifications on function. (a) A c-mycepitope tag appended to the KCC1 N terminus does not affect function (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 (337) 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 (456) Google Scholar). (b) Interchanging N termini between sNKCC1 and hNKCC1 has no effect on ion affinity differences; rather the dissimilarities are conferred by the large central hydrophobic domains (21Isenring P. Forbush III, B. J. Biol. Chem. 1997; 272: 24556-24562Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar).A significant finding of this report is the activation of h1r2A0.7 in response to a decrease in [Cl]. This suggests that NKCC2 is modulated by [Cl]i in the renal epithelial cell, providing a way for the apical cotransporter to respond to changes in the rate of Cl exit across the basolateral membrane. In many species vasopressin regulates NaCl and water reabsorption in the medullary thick ascending limb via a cAMP-dependent mechanism. By demonstrating a route for basolateral → apical communication, the present result supports a model in which the initial point of cAMP regulation is at the basolateral exit pathways (33Schlatter E. Greger R. Pfluegers Arch. Eur. J. Physiol. 1985; 405: 367-376Crossref PubMed Scopus (87) Google Scholar), although it does not rule out a model in which the cotransporter is the primary site of regulation (34Molony D.A. Reeves W.B. Hebert S.C. Andreoli T.E. Am. J. Physiol. 1987; 252: F177-F187Crossref PubMed Google Scholar).Compared with NKCC1, h1r2A0.7 displays a higher level of constitutive activity and exhibits a faster and smaller response to changes in intracellular Cl (see Fig. 6). Together, the results suggest that the [Cl]i set point is higher for NKCC2. In addition, the h1r2A0.7 response to [Cl]i appears to be more easily overridden by a response to a change in cell volume.Our previous results with sNKCC1/hNKCC1 chimeras demonstrate that the N- and C termini of NKCC are not important in determining ion and bumetanide affinity differences, and it is reasonable to expect that the chimera h1r2A0.7 is fully representative of NKCC2A with regard to these properties. On the other hand, the N terminus of sNKCC1 has been shown to be involved in regulation of transport by a mechanism that involves phosphorylation of T184 and T189 (17Lytle C. Forbush III, B. J. Biol. Chem. 1992; 267: 25438-25443Abstract Full Text PDF PubMed Google Scholar, 35Behnke R. Forbush B. Bull. Mt. Desert Is. Bio. Lab. 1997; 36: 59Google Scholar). The 15-residue region surrounding these phosphoacceptors is well conserved, with a single amino acid change in the h1r2A0.7 chimera (Q98(NKCC2) → R183(NKCC1)). We do not know if the region upstream of the phosphoacceptors plays a role in regulation and, if so, whether the introduction of NKCC1 residues in this region makes the regulatory behavior of h1r2A0.7 different from that of native NKCC2A.The HEK cell cotransporter has functional characteristics that are quite different from both NKCC1 and h1r2A0.7. In particular, the affinity of the endogenous transporter for Rb is 8- and 2.5-fold lower than that of NKCC1 and h1r2A0.7, respectively, and the affinity for Na is >1.5-fold lower than that of both of the described isoforms. The results of RT-PCR experiments demonstrate that the HEK cell does not express a detectable message for NKCC2 and that NKCC1, or a form homologous to NKCC1, is present (Fig. 7).We consider three possible explanations for the uniqueness of the HEK cell cotransporter. (a) Assuming that the endogenous HEK cell cotransporter is in fact hNKCC1, why is the kinetic and regulatory behavior different from hNKCC1? One possibility is that the HEK cell cotransporter contains an accessory subunit that is not available in the amounts necessary to accompany overexpressed hNKCC1 in transfected cells. A related idea is that the hNKCC1 protein may be post-translationally modified and that the modification machinery is inadequate for the overexpressed protein. (b) It is possible that the HEK cell cotransporter is a splice variant of hNKCC1, with functional characteristics that are different from the transporter encoded by the cDNA isolated from T-84 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 (224) Google Scholar). We have reported three splice variants of NKCC2 that differ in the sequence of the predicted second transmembrane domain. Although the hypothesis has not yet been tested, alternative splicing of this region might be expected to result in transporters with different ion affinities (9Payne J.A. Forbush III, B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4544-4548Crossref PubMed Scopus (254) Google Scholar). In a search for splice variants of NKCC1, Delpire and co-workers (36Randall, J., Thorne, T., and Delpire, E. (19987) Am. J. Physiol., in press.Google Scholar) found no evidence of alternative splicing in this region but did report that 16 residues in the C terminus are sometimes removed by splicing. In light of our recent finding that sNKCC1/hNKCC1 ion affinity differences are due only to differences within the central hydrophobic domain (21Isenring P. Forbush III, B. J. Biol. Chem. 1997; 272: 24556-24562Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), it seems unlikely that this alteration would result in the discrepancy between HEK cell affinities and those of hNKCC1. (c) It is possible that the HEK cell cotransporter is an isoform of NKCC that has not yet been identified. At present there is no direct evidence for or against this hypothesis. However, in a general sense, the possibility of additional isoforms is supported by the broad distribution of the Na-K-Cl cotransporter in cells with very different physiological function. In epithelial tissues, the function of the Na-K-Cl cotransporter is transepithelial transport, and regulation of the transporter is presumably optimized for hormonal control and/or apical-basolateral communication. In non-polarized tissues, the Na-K-Cl cotransporter plays a role in regulation of intracellular volume (1Haas M. Annu. Rev. Physiol. 1989; 51: 443-457Crossref PubMed Scopus (173) Google Scholar) and may also be important in its effect on extracellular [K] (37McManus T.J. Haas M. Starke L.C. Lytle C.Y. Ann. N. Y. Acad. Sci. 1985; 456: 183-186Crossref PubMed Scopus (36) Google Scholar). Thus, we propose that the HEK cell cotransporter may represent a unique NKCC isoform that operates with low cation affinities. 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. 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 (173) 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 (173) 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 (337) 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 (337) 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 (456) 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 (224) 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 (254) 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 (158) Google Scholar, 13Ecelbarger C.A. Terris J. Hoyer J.R. Nielsen S. Wade J.B. Knepper M.A. Am. J. 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A. 1993; 90: 2749-2753Crossref PubMed Scopus (338) 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 (173) 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" @default.
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