A Dominant Negative Mutant of the KCC1 K-Cl Cotransporter

K-Cl cotransport regulates cell volume and chloride equilibrium potential. Inhibition of erythroid K-Cl cotransport has emerged as an important adjunct strategy for the treatment of sickle cell anemia. However, structure-function relationships among the polypeptide products of the four K-Cl cotransporter (KCC) genes are little understood. We have investigated the importance of the N- and C-terminal cytoplasmic domains of mouse KCC1 to its K-Cl cotransport function expressed in Xenopus oocytes. Truncation of as few as eight C-terminal amino acids (aa) abolished function despite continued polypeptide accumulation and surface expression. These C-terminal loss-of-function mutants lacked a dominant negative phenotype. Truncation of the N-terminal 46 aa diminished function. Removal of 89 or 117 aa (ΔN117) abolished function despite continued polypeptide accumulation and surface expression and exhibited dominant negative phenotypes that required the presence of the C-terminal cytoplasmic domain. The dominant negative loss-of-function mutant ΔN117 was co-immunoprecipitated with wild type KCC1 polypeptide, and its co-expression did not reduce wild type KCC1 at the oocyte surface. ΔN117 also exhibited dominant negative inhibition of human KCC1 and KCC3 and, with lower potency, mouse KCC4 and rat KCC2. K-Cl cotransport regulates cell volume and chloride equilibrium potential. Inhibition of erythroid K-Cl cotransport has emerged as an important adjunct strategy for the treatment of sickle cell anemia. However, structure-function relationships among the polypeptide products of the four K-Cl cotransporter (KCC) genes are little understood. We have investigated the importance of the N- and C-terminal cytoplasmic domains of mouse KCC1 to its K-Cl cotransport function expressed in Xenopus oocytes. Truncation of as few as eight C-terminal amino acids (aa) abolished function despite continued polypeptide accumulation and surface expression. These C-terminal loss-of-function mutants lacked a dominant negative phenotype. Truncation of the N-terminal 46 aa diminished function. Removal of 89 or 117 aa (ΔN117) abolished function despite continued polypeptide accumulation and surface expression and exhibited dominant negative phenotypes that required the presence of the C-terminal cytoplasmic domain. The dominant negative loss-of-function mutant ΔN117 was co-immunoprecipitated with wild type KCC1 polypeptide, and its co-expression did not reduce wild type KCC1 at the oocyte surface. ΔN117 also exhibited dominant negative inhibition of human KCC1 and KCC3 and, with lower potency, mouse KCC4 and rat KCC2. cation chloride cotransporter K-Cl cotransporter N-ethylmaleimide polymerase chain reaction N-terminally truncated C-terminally truncated mouse KCC1 human KCC1 reverse transcriptase immunoprecipitation phosphate-buffered saline wild type amino acid(s) Secondary active transport of chloride across cell plasma membranes is achieved by ion symport and antiport mechanisms. The major chloride symporters are members of the phylogenetically ancient CCC1 cation chloride cotransporter superfamily, comprising in mammals two NKCC genes encoding multiple bumetanide-sensitive Na-K-2Cl cotransporter polypeptides (1Russell J.M. Physiol. Rev. 2000; 80: 211-276Crossref PubMed Scopus (737) Google Scholar, 2Haas M. Forbush B. Annu. Rev. Physiol. 2000; 62: 515-534Crossref PubMed Scopus (332) Google Scholar), one NCC gene encoding thiazide-sensitive NaCl cotransporter polypeptides (3Monroy A. Plata C. Hebert S.C. Gamba G. Am. J. Physiol. 2000; 279: F161-F169Crossref PubMed Google Scholar), and at least four KCC encoding multiple K-Cl cotransporter polypeptides (4Lauf P.K. Adragna N.C. Cell Physiol. Biochem. 2000; 10: 341-354Crossref PubMed Scopus (193) Google Scholar). K-Cl cotransporters in most cell types mediate solute efflux and regulatory volume decrease, opposing the solute import functions of NKCCs. K-Cl cotransporters also serve to regulate the cellular electrochemical equilibrium potential for Cl− and can regulate [K+] of the interstitial space, especially in the nervous system. KCC1 cDNAs have been cloned from rabbits (5Gillen C.M. Brill S. Payne J.A. Forbush B. J. Biol. Chem. 1996; 271: 16237-16244Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar), rats (5Gillen C.M. Brill S. Payne J.A. Forbush B. J. Biol. Chem. 1996; 271: 16237-16244Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar), humans (5Gillen C.M. Brill S. Payne J.A. Forbush B. J. Biol. Chem. 1996; 271: 16237-16244Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar), pigs (6Holtzman E.J. Kumar S. Faaland C.A. Warner F. Logue P.J. Erickson S.J. Ricken G. Wladman J. Kumar S. Dunham P.B. Am. J. Physiol. 1998; 275: F550-F564PubMed Google Scholar), and mice (7Su W. Shmukler B.E. Chernova M.N. Stuart-Tilley A.K. De Franceschi L. Brugnara C. Alper S.L. Am. J. Physiol. 1999; 277: C899-C912Crossref PubMed Google Scholar, 8Pellegrino C.M. Rubicki A.C. Musto S. Nagel R.L. Schwartz R.S. Blood Cells Mol. Dis. 1998; 24: 31-40Crossref PubMed Scopus (65) Google Scholar), with gene structures reported for humans (6Holtzman E.J. Kumar S. Faaland C.A. Warner F. Logue P.J. Erickson S.J. Ricken G. Wladman J. Kumar S. Dunham P.B. Am. J. Physiol. 1998; 275: F550-F564PubMed Google Scholar), mice (9Shmukler B.E. Brugnara C. Alper S.L. Biochim. Biophys. Acta. 2000; 1492: 353-361Crossref PubMed Scopus (9) Google Scholar), and Caenorhabditis elegans (6Holtzman E.J. Kumar S. Faaland C.A. Warner F. Logue P.J. Erickson S.J. Ricken G. Wladman J. Kumar S. Dunham P.B. Am. J. Physiol. 1998; 275: F550-F564PubMed Google Scholar). KCC2 cDNA has been cloned from rat (10Payne J.A. Stevenson T.J. Donaldson L.F. J. Biol. Chem. 1996; 271: 16245-16252Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar). Two isoforms of human KCC3 cDNA (11Hiki K. D'Andrea R.J. Furze J. Crawford J. Woollatt E. Sutherland G.R. Vadas M.A. Gamble J.R. J. Biol. Chem. 1999; 274: 10661-10667Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 12Race J.E. Makhlouf F.N. Logue P.J. Wilson F.H. Dunham P.B. Holtzman E.J. Am. J. Physiol. 1999; 277: C1210-C1219Crossref PubMed Google Scholar, 13Mount D.B. Mercado A. Song L. Xu J. George A.L. Delpire E. Gamba G. J. Biol. Chem. 1999; 274: 16355-16362Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar), KCC4 cDNA from humans and mice (13Mount D.B. Mercado A. Song L. Xu J. George A.L. Delpire E. Gamba G. J. Biol. Chem. 1999; 274: 16355-16362Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar), and a Xenopusoocyte partial cDNA encoding a KCC isoform of the KCC1/3 family (14Mercado A. de los Heros P. Vazquez N. Meade P. Mount D.B. Gamba G. Am. J. Physiol. 2001; 261: C670-C680Crossref Google Scholar) have also been reported. NKCCs are activated by hypertonic cell shrinkage and by activators of protein phosphorylation. NKCCs tend to be inhibited by cell swelling, by sulfhydryl oxidizing agents, and by inhibitors of protein dephosphorylation. In contrast, KCCs are activated by hypotonic cell swelling, by sulfhydryl oxidizing agents, and by inhibitors of protein phosphorylation. KCCs tend to be inhibited by cell shrinkage and by inhibitors of protein dephosphorylation (4Lauf P.K. Adragna N.C. Cell Physiol. Biochem. 2000; 10: 341-354Crossref PubMed Scopus (193) Google Scholar). Heterologous overexpression of KCC1 leads to up-regulation of endogenous NKCC activity (15Gillen C.M. Forbush B. Am. J. Physiol. 1999; 276: C328-C336Crossref PubMed Google Scholar). The molecular mechanisms by which KCCs are regulated are unknown. The transmembrane region of CCC polypeptides, based on data from NKCC1, probably spans the lipid bilayer 12 times (16Gerelsaikhan T. Turner R.J. J. Biol. Chem. 2000; 275: 40471-40477Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Transmembrane helices 2, 4, and 7 of NKCC1 have been implicated in ion binding by site-directed mutagenesis studies (17Isenring P. Jacoby S.C. Chang J. Forbush B. J. Gen. Physiol. 1998; 112: 549-558Crossref PubMed Scopus (78) Google Scholar). The two initial reports on structure-function relationships of K-Cl cotransporters concern aspects of their C-terminal cytoplasmic tails. Tyr1087 of rat KCC2, close to KCC2's C terminus, and the analogous residue in rabbit KCC1 are each required for hypotonic activation of ion transport activity in Xenopusoocytes. However, neither residue is required for delivery to the oocyte surface or for inhibition of hypotonically activated transport activity by serine-threonine phosphatase inhibitors (18Strange K. Singer T.D. Morrison R. Delpire E. Am. J. Physiol. 2000; 279: C860-C867Crossref PubMed Google Scholar). In P.K. J. Delpire E. Adragna N.C. Physiol. Biochem. 2001; PubMed Scopus Google Scholar) have that from rabbit KCC1 of most of the C-terminal cytoplasmic abolished activation by N-ethylmaleimide in not surface expression. Inhibition of the efflux cell shrinkage has an important to the of sickle cell The major efflux of the sickle are K-Cl cotransport and the C. De Franceschi L. N. M. A. Alper S.L. N. PubMed Scopus Google Scholar). inhibitors of the erythroid are C. B. C. S. M. N. Alper S.L. J. 1996; PubMed Scopus Google Scholar), and in continued inhibitors of K-Cl cotransport have not been KCC inhibitors serve as an adjunct treatment of sickle cell as to with of Franceschi L. F. G. T. Alper S.L. Brugnara C. J. PubMed Scopus Google Scholar). KCC inhibitors also a to the of K-Cl cotransport in cell function. We have structure-function studies with the mouse KCC1 K-Cl We that the C-terminal cytoplasmic and the of the N-terminal cytoplasmic are required for transport function in Xenopus oocytes. In that of the N-terminal cytoplasmic from KCC1 a dominant negative that the presence of the C-terminal cytoplasmic domain. The dominant negative mutant polypeptide with the wild type KCC1 polypeptide and its dominant negative with KCC gene dominant negative mutant on the of of KCC K-Cl of encoding wild type cDNA (7Su W. Shmukler B.E. Chernova M.N. Stuart-Tilley A.K. De Franceschi L. Brugnara C. Alper S.L. Am. J. Physiol. 1999; 277: C899-C912Crossref PubMed Google Scholar) was to in a reaction volume of the in the at for which the The reaction for at and for at for at and for at of at was by to products in from with the and cloned the of the cloned products was with an was with the of of the of mouse KCC1 by the amino acids of the KCC1 N-terminal cytoplasmic The and of was for in with the reverse (7Su W. Shmukler B.E. Chernova M.N. Stuart-Tilley A.K. De Franceschi L. Brugnara C. Alper S.L. Am. J. Physiol. 1999; 277: C899-C912Crossref PubMed Google Scholar). encoding each mutant by a of the encoding the of and encoding the Xenopus oocyte expression of (7Su W. Shmukler B.E. Chernova M.N. Stuart-Tilley A.K. De Franceschi L. Brugnara C. Alper S.L. Am. J. Physiol. 1999; 277: C899-C912Crossref PubMed Google Scholar). The mutants are in mutant is by in which the mutant wild type and wild type of C-terminal mutant (7Su W. Shmukler B.E. Chernova M.N. Stuart-Tilley A.K. De Franceschi L. Brugnara C. Alper S.L. Am. J. Physiol. 1999; 277: C899-C912Crossref PubMed Google Scholar) was for in with reverse a in of the encoding the was with and with the of mutants by at a the region for for for or for and at the was and by These the C-terminal amino acids to the to to and to and to is by in which the wild type amino to the is The mutant was by of the of ΔN117 with the of KCC2 cDNA (10Payne J.A. Stevenson T.J. Donaldson L.F. J. Biol. Chem. 1996; 271: 16245-16252Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar) was the of J. Payne of KCC3 cDNA J.E. Makhlouf F.N. Logue P.J. Wilson F.H. Dunham P.B. Holtzman E.J. Am. J. Physiol. 1999; 277: C1210-C1219Crossref PubMed Google Scholar) was the of P. Dunham and E. Holtzman KCC4 cDNA was cloned by from mouse The was from The was (7Su W. Shmukler B.E. Chernova M.N. Stuart-Tilley A.K. De Franceschi L. Brugnara C. Alper S.L. Am. J. Physiol. 1999; 277: C899-C912Crossref PubMed Google Scholar), encoding the C-terminal amino in KCC1 and KCC4 KCC1 was cloned by from as and (7Su W. Shmukler B.E. Chernova M.N. Stuart-Tilley A.K. De Franceschi L. Brugnara C. Alper S.L. Am. J. Physiol. 1999; 277: C899-C912Crossref PubMed Google Scholar). The of the and cDNAs was by encoding the was from cell by as and (7Su W. Shmukler B.E. Chernova M.N. Stuart-Tilley A.K. De Franceschi L. Brugnara C. Alper S.L. Am. J. Physiol. 1999; 277: C899-C912Crossref PubMed Google Scholar). The was the by and cDNAs the oocyte expression (7Su W. Shmukler B.E. Chernova M.N. Stuart-Tilley A.K. De Franceschi L. Brugnara C. Alper S.L. Am. J. Physiol. 1999; 277: C899-C912Crossref PubMed Google Scholar). with polymerase from and In of polypeptide with was with the rabbit in the presence of membranes in a reaction was The reaction was and by the of of immunoprecipitation and was as (7Su W. Shmukler B.E. Chernova M.N. Stuart-Tilley A.K. De Franceschi L. Brugnara C. Alper S.L. Am. J. Physiol. 1999; 277: C899-C912Crossref PubMed Google Scholar). Xenopus with to partial for with at in type in and with of at the or the with of or or the of in at for with of M.N. L. M. M. K. Alper S.L. J. Gen. Physiol. PubMed Scopus Google Scholar), for ion transport function was by of of in The was by of of of was by at in of was in a of for of and to endogenous NKCC and or which chloride for with was was at expressed as of and as for each oocytes. a of from of oocyte expression of wild type of hypotonically activated of of the the of hypotonic was in and by was in by with for the a at in oocyte and and of at the was at in a was by or was to with rabbit to N-terminal aa and to C-terminal aa and by (7Su W. Shmukler B.E. Chernova M.N. Stuart-Tilley A.K. De Franceschi L. Brugnara C. Alper S.L. Am. J. Physiol. 1999; 277: C899-C912Crossref PubMed Google Scholar). with of and with or of at in oocyte NaCl in a with a and to of and of in a at to NaCl and with rabbit at with the presence of for and with protein at times in of NaCl and times in of NaCl and by M.N. Alper S.L. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). The immunoprecipitation of wild type KCC1 and ΔN117 KCC1 in in the presence of of at to in phosphate-buffered saline and for in the with was with and was continued for a The and for in in of in and a the by The with for with The times in NaCl and times in NaCl and with on a to and to or in each with in of for at with to for and for in with and with was by in with and in in for and in 1999; PubMed Google Scholar). a or two of KCC1 a and with the from at least two of for each KCC1 of with expressed in Xenopus was an of by hypotonic the of the of was in was in in studies of in Xenopus (7Su W. Shmukler B.E. Chernova M.N. Stuart-Tilley A.K. De Franceschi L. Brugnara C. Alper S.L. Am. J. Physiol. 1999; 277: C899-C912Crossref PubMed Google Scholar) and studies of rabbit (5Gillen C.M. Brill S. Payne J.A. Forbush B. J. Biol. Chem. 1996; 271: 16237-16244Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, P.K. J. Delpire E. Adragna N.C. Physiol. Biochem. 2001; PubMed Scopus Google Scholar) and human KCC1 in (6Holtzman E.J. Kumar S. Faaland C.A. Warner F. Logue P.J. Erickson S.J. Ricken G. Wladman J. Kumar S. Dunham P.B. Am. J. Physiol. 1998; 275: F550-F564PubMed Google Scholar) and of Xenopus oocyte K-Cl cotransport (14Mercado A. de los Heros P. Vazquez N. Meade P. Mount D.B. Gamba G. Am. J. Physiol. 2001; 261: C670-C680Crossref Google Scholar) have that activated chloride and is inhibited by the serine-threonine phosphatase inhibitors and and by the Removal of the eight C-terminal from the C-terminal cytoplasmic of as in a of activity in abolished of by of the C-terminal cytoplasmic residue or abolished activity of with in wild type was abolished by of the C-terminal eight amino acids and by of the C-terminal These mutant polypeptides in Xenopus to lower for polypeptide to to and with cytoplasmic N-terminal aa or cytoplasmic C-terminal aa of wild type at or the cell surface was in and was in However, that and expression at or the oocyte surface despite their of transport activity The of in with with the of polypeptide on did not at the oocyte surface despite its accumulation the and of in the data a expressed The presence of the was in human from and in and with two In of a polypeptide in which of is by amino at is for the exhibited transport function in However, of was by in mouse and The of the N-terminal 46 of to from that of in 4, exhibited diminished hypotonic activation of wild type of with hypotonically wild type or for with for N-terminal of 89 or 117 (ΔN117) abolished activation by The by was not for hypotonic and activity also abolished by N-terminal of mutant polypeptides to lower wild type However, polypeptides at or the oocyte surface at to that of wild type as by with to N-terminally truncated polypeptide We the that among the loss-of-function mutants by of C-terminal and N-terminal cytoplasmic be one or that a dominant negative phenotype. type and in and for transport function and for surface expression. that expressed in a of did not by wild type was of mutant and wild type polypeptides expressed at or the cell and mutant co-expression did not reduce the of wild type at the oocyte of with wild type and with one of the mutants or at to wild type We also the that co-expression of and mutants function. However, co-expression of ΔN117 with did not by of the mutant ΔN117 with an of wild type by 7 of the mutant with an of wild type a in activity of wild type polypeptide at or the oocyte surface was not diminished by co-expression of at or the surface at 7 7 C that KCC1 and ΔN117 KCC1 at the oocyte surface in and surface polypeptide a of oocyte with and with the in 7 for ΔN117 in oocyte and and in from and the presence in oocyte of with that of which is lower in the These are from the protein in the of neither KCC1 was by co-expression of ΔN117 KCC1 and dominant negative of KCC1 function by ΔN117 KCC1 is not achieved by of KCC1 expression or surface of dominant negative of K-Cl cotransport by ΔN117 of ΔN117 inhibition of of was at a oocytes. are as among of are by a with are by at the ΔN117 function of wild type in a at of expressed at of ΔN117 function of wild type the did not wild type function at a the for neither was in the mutant the of dominant negative exhibited by mutant a for the C-terminal cytoplasmic or little accumulation of mutant of the dominant negative was also by of of by transport for was by co-expression of of two loss-of-function mutants of an the mutant polypeptide oocytes. The human mutant a of dominant not at the surface of the cell or of the Xenopus oocyte and not a dominant negative Franceschi L. F. G. T. Alper S.L. Brugnara C. J. PubMed Scopus Google Scholar). not activity at a In contrast, the mouse mutant in the of is expressed at the oocyte surface 1999; PubMed Google Scholar). is for did not The dominant negative of ΔN117 as as its co-expression with wild type at the oocyte that the mutant and the wild type polypeptides in the was by in in wild type was by and and In contrast, ΔN117 was by and ΔN117 not in the also in the despite the of the N-terminal in the ΔN117 of and ΔN117 was not in was in the of or polypeptides in and of and ΔN117 was also in In from with encoding ΔN117 and wild type polypeptides, the is for in polypeptides, the also polypeptides, its was not in one of ΔN117 and wild type KCC1 polypeptides in the Xenopus oocyte and their The negative loss-of-function mutant was not co-immunoprecipitated with wild type by despite the presence of the truncated polypeptide by in of and oocytes. the mutant did not with in from of The KCC gene family has two one by the KCC1 and KCC3 and the comprising KCC2 and KCC4 to (4Lauf P.K. Adragna N.C. Cell Physiol. Biochem. 2000; 10: 341-354Crossref PubMed Scopus (193) Google Scholar, A. Song L. Vazquez N. Mount D.B. Gamba G. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). cell types can one KCC gene the of a dominant negative KCC1 to K-Cl cotransporter activity in on its to K-Cl cotransport activity by the polypeptide products of KCC that ΔN117 at hypotonically activity of wild type as as that of wild type also hypotonically activity of that hypotonically activity of wild type also was by KCC1 and KCC4 activity was inhibited at a and at a wild of The activity of in Xenopus has been A. Song L. Vazquez N. Mount D.B. Gamba G. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). C that hypotonically activity of was also inhibited by ΔN117 and wild of to and of KCC2 transport activity in hypotonic of ΔN117 also to inhibition in activity of KCC2 in has been (18Strange K. Singer T.D. Morrison R. Delpire E. Am. J. Physiol. 2000; 279: C860-C867Crossref PubMed Google Scholar, J.A. Am. J. Physiol. PubMed Google Scholar). We have structure-function of the N-terminal and C-terminal cytoplasmic of the KCC1 K-Cl We have that of of cytoplasmic to Xenopus by or by C-terminal exhibited loss-of-function phenotypes of accumulation at or the oocyte dominant negative Removal of the N-terminal 89 or 117 amino acids from also loss-of-function mutants that wild type of expression at or the oocyte surface. The ΔN117 mutant to be a dominant negative of wild type ion transport function not wild type polypeptide at the oocyte surface. The ΔN117 mutant and wild type polypeptides in from in which or from in in the presence of In contrast, the negative loss-of-function mutant polypeptide did not with wild type polypeptide in dominant negative of wild type transport function by ΔN117 and the of ΔN117 to with wild type polypeptide required of the C-terminal residue ΔN117 also exhibited dominant negative inhibition of and, of by and The of the C-terminal for of by or by on the importance of rabbit KCC1 the analogous rat KCC2 Tyr1087 to hypotonic (18Strange K. Singer T.D. Morrison R. Delpire E. Am. J. Physiol. 2000; 279: C860-C867Crossref PubMed Google Scholar). also and reported for of rabbit KCC1 in P.K. J. Delpire E. Adragna N.C. Physiol. Biochem. 2001; PubMed Scopus Google Scholar). We have that of as few as eight C-terminal amino acids from abolished function in surface expression. C-terminal mutant polypeptides in mutants expressed at at the cell surface. and of in the data the expressed In of a polypeptide in which of is by amino at The is in human from and In with polypeptide exhibited transport function in Xenopus with C-terminal are to at least one In to the for the C-terminal of 46 N-terminal from partial hypotonic of by did not However, of 89 or 117 N-terminal abolished by These are the data a required for the N-terminal cytoplasmic in KCC function. The N-terminal cytoplasmic to be for delivery to and accumulation of at the oocyte surface. The N-terminal cytoplasmic of NKCC1 a binding for protein phosphatase which NKCC1 aa to ion transport A. Forbush B. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). is from cytoplasmic of However, are the N-terminal cytoplasmic region by as for by or by Two the that as a in Xenopus oocytes. the ΔN117 mutant of ion transport function of wild type the mutant and wild type polypeptides in and in can be co-immunoprecipitated by that wild type has been for rat NKCC1 based on Turner R.J. 2000; PubMed Scopus Google Scholar). and also of as as of KCC gene A. S. and S. L. an of a polypeptide from and not in ΔN117 is the reported dominant negative among KCC K-Cl cotransporters and the dominant negative among However, two of dominant negative have been reported. The is the of mouse the of two C-terminal polypeptide D.B. A. Plata C. Xu Gamba G. Hebert S.C. Am. J. Physiol. 1999; 276: PubMed Google Scholar, C. Mount D.B. Hebert S.C. Gamba G. Am. J. Physiol. 1999; 276: PubMed Google Scholar). of polypeptide with the and active of inhibited its cation transport activity in a that was by C. Mount D.B. Hebert S.C. Gamba G. Am. J. Physiol. 1999; 276: PubMed Google Scholar). and isoforms of are in mouse of A. Forbush B. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar), was to the of to activity in of the mouse C. Mount D.B. Hebert S.C. Gamba G. Am. J. Physiol. 1999; 276: PubMed Google Scholar). dominant negative of CCC was of a CCC cDNA in the expressed data and amino with NKCC1 L. F. E. P. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). expressed in and to the cell is as a of or inhibited NKCC1 lacked dominant negative activity with the or with in of one be co-immunoprecipitated with endogenous NKCC1 The of inhibited activity in with of inhibited the was C. Mount D.B. Hebert S.C. Gamba G. Am. J. Physiol. 1999; 276: PubMed Google Scholar). of and NKCC1 to inhibition of NKCC1 function L. F. E. P. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). Inhibition by ΔN117 of wild type and in 7 and was of or of mutant and wild type transport protein in is based on of that the of of the polypeptides at the cell surface or and for and These are to be for a of R. PubMed Scopus Google Scholar) and dominant negative of N. 1999; PubMed Scopus Google Scholar, L. C. M. Schwartz P.J. Am. J. Physiol. 2000; 279: PubMed Google Scholar) for the of ΔN117 KCC1 with KCC1 or for dominant negative CCC in expression the data in with the of a of and R. PubMed Scopus Google Scholar) not and of the of data a not and that wild type KCC polypeptide gene products are at least with the of NKCC1 Turner R.J. 2000; PubMed Scopus Google Scholar). In each mutant in the can be to the A. S. B. E. and S. L. the presence of neither cytoplasmic of is required for The of ΔN117 to ion transport function of and to and that the products of the KCC genes can to polypeptides with the ion regulatory and to inhibitors among the KCC gene products (4Lauf P.K. Adragna N.C. Cell Physiol. Biochem. 2000; 10: 341-354Crossref PubMed Scopus (193) Google Scholar, 12Race J.E. Makhlouf F.N. Logue P.J. Wilson F.H. Dunham P.B. Holtzman E.J. Am. J. Physiol. 1999; 277: C1210-C1219Crossref PubMed Google Scholar, A. Song L. Vazquez N. Mount D.B. Gamba G. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, J.A. Am. J. Physiol. PubMed Google Scholar), of also in be especially for regulatory that of and inhibitors of KCC K-Cl cotransporters are in KCC isoform expression have been P. Brill S. Forbush B. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). of the functions of KCC polypeptides and the of of K-Cl cotransport activity or These of or expression of dominant negative or KCC K-Cl cotransporters are expressed among and cell cell types probably one KCC gene the of of a of one gene by or expression of genes is a In overexpression of ΔN117 or mutants a for inhibition of or most KCC gene We P. Dunham and J. Payne for the of cDNA