Self-interacting Domains in the C Terminus of a Cation-Cl- Cotransporter Described for the First Time

The first isoform of the Na+-K+-Cl- cotransporter (NKCC1), a widely distributed member of the cation-Cl- cotransporter superfamily, plays key roles in many physiological processes by regulating the ion and water content of animal cells and by sustaining electrolyte secretion across various epithelia. Indirect studies have led to the prediction that NKCC1 operates as a dimer assembled through binding domains that are distal to the amino portion of the carrier. In this study, evidence is presented that NKCC1 possesses self-interacting properties that result in the formation of a large complex between the proximal and the distal segment of the cytosolic C terminus. Elaborate mapping studies of these segments showed that the contact sites are dispersed along the entire C terminus, and they also led to the identification of a critical interacting residue that belongs to a putative forkhead-associated binding domain. In conjunction with previous findings, our results indicate that the uncovered interacting domains are probably a major determinant of the NKCC1 conformational landscape and assembly into a high order structure. A model is proposed in which the carrier could alternate between monomeric and homo-oligomeric units via chemical- or ligand-dependent changes in conformational dynamics. The first isoform of the Na+-K+-Cl- cotransporter (NKCC1), a widely distributed member of the cation-Cl- cotransporter superfamily, plays key roles in many physiological processes by regulating the ion and water content of animal cells and by sustaining electrolyte secretion across various epithelia. Indirect studies have led to the prediction that NKCC1 operates as a dimer assembled through binding domains that are distal to the amino portion of the carrier. In this study, evidence is presented that NKCC1 possesses self-interacting properties that result in the formation of a large complex between the proximal and the distal segment of the cytosolic C terminus. Elaborate mapping studies of these segments showed that the contact sites are dispersed along the entire C terminus, and they also led to the identification of a critical interacting residue that belongs to a putative forkhead-associated binding domain. In conjunction with previous findings, our results indicate that the uncovered interacting domains are probably a major determinant of the NKCC1 conformational landscape and assembly into a high order structure. A model is proposed in which the carrier could alternate between monomeric and homo-oligomeric units via chemical- or ligand-dependent changes in conformational dynamics. Cation-Cl- cotransporters (CCCs) 1The abbreviations used are: CCC, cation-Cl- cotransporter; KCC, K+-Cl- cotransporter; NCC, Na+-Cl- cotransporter; NKCC, Na+-K+-Cl- cotransporter; ADH, anti-diuretic hormone; AMPR, ampicilline resistance gene; del, truncated; GST, glutathione S-transferase, GS, glutathione-coupled Sepharose; HA, hemagglutinin; hu, human; MCS, multiple cloning site; op, operator; Pn, position; Y, EGY48 yeast; Yop, EGY48 yeast transformed with the p8op-lacZ reporter plasmid; Ab, antibody; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. belong to an important family of transport systems that couple the movement of Cl- to that of Na+ and/or K+ across cell surfaces (1Haas M. Forbush B. Annu. Rev. Physiol. 2000; 62: 515-534Crossref PubMed Scopus (331) Google Scholar, 2Isenring P. Forbush B. Comp. Biochem. Physiol. Part A Mol. Integr. Physiol. 2001; 130: 487-497Crossref PubMed Scopus (59) Google Scholar, 3Payne J.A. Forbush B. Curr. Opin. Cell Biol. 1995; 7: 493-503Crossref PubMed Scopus (108) Google Scholar, 4Russell J.M. Physiol. Rev. 2000; 80: 211-276Crossref PubMed Scopus (734) Google Scholar). One of the first CCCs identified was the secretory Na+-K+-Cl- cotransporter (5Xu J.C. Lytle C. Zhu T.T. Payne J.A. Benz E. Forbush B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2201-2205Crossref PubMed Scopus (370) Google Scholar), also called NKCC1. Since then, six other family members have been identified: the renal Na+-K+-Cl- cotransporters (6Payne J.A. Forbush B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4544-4548Crossref PubMed Scopus (258) Google Scholar), the Na+-Cl- cotransporter (7Gamba 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 (342) Google Scholar), and the K+-Cl- cotransporters (8Gillen C.M. Brill S. Payne J.A. Forbush B. J. Biol. Chem. 1996; 271: 16237-16244Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 9Mount 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 (249) Google Scholar, 10Payne J.A. Stevenson T.J. Donaldson L.F. J. Biol. Chem. 1996; 271: 16245-16252Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar, 11Race 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) called NKCC2, NCC, and KCCs, respectively. The CCC family also includes carrier-like molecules for which no ionic substrates have been identified to date; they are called orphan CCCs (12Caron L. Rousseau F. Gagnon E. Isenring P. J. Biol. Chem. 2000; 275: 32027-32036Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The main function of a CCC is to regulate the ion or volume content of individual cells and to facilitate salt movement across various epithelia (1Haas M. Forbush B. Annu. Rev. Physiol. 2000; 62: 515-534Crossref PubMed Scopus (331) Google Scholar, 4Russell J.M. Physiol. Rev. 2000; 80: 211-276Crossref PubMed Scopus (734) Google Scholar); CCCs may play accessory roles in acid base balance or regulation of cell pH by transporting NH +4 at the K+ site or by modulating intracellular [Cl-] (13Bergeron M.J. Gagnon E. Wallendorff B. Lapointe J.Y. Isenring P. Am. J. Physiol. 2003; 285: F68-F78Crossref PubMed Scopus (51) Google Scholar, 14Boettger T. Hubner C.A. Maier H. Rust M.B. Beck F.X. Jentsch T.J. Nature. 2002; 25: 874-878Crossref Scopus (339) Google Scholar). The activity of these proteins is tightly modulated through interactions with regulatory enzymes (15Darman R.B. Flemmer A. Forbush B. J. Biol. Chem. 2001; 276: 34359-34362Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 16Darman R.B. Forbush B. J. Biol. Chem. 2002; 277: 37542-37550Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 17Dowd B.F. Forbush B. J. Biol. Chem. 2003; 278: 27347-27353Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 18Flemmer A.W. Gimenez I. Dowd B.F. Darman R.B. Forbush B. J. Biol. Chem. 2002; 277: 37551-37558Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 19Piechotta K. Lu J. Delpire E. J. Biol. Chem. 2002; 277: 50812-50819Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 20Piechotta K. Garbarini N. England R. Delpire E. J. Biol. Chem. 2003; 278: 52848-52856Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar) and with cytoskeletal elements (21D'Andrea L. Lytle C. Matthews J.B. Hofman P. Forbush B. Madara J.L. J. Biol. Chem. 1996; 271: 28969-28976Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 22Liedtke C.M. Hubbard M. Wang X. Am. J. Physiol. 2003; 284: C487-C496Crossref PubMed Scopus (58) Google Scholar). The best example studied thus far is the phosphorylation-dependent activation of NKCC1, a ubiquitous CCC that is expressed basolaterally in polarized cells (1Haas M. Forbush B. Annu. Rev. Physiol. 2000; 62: 515-534Crossref PubMed Scopus (331) Google Scholar, 2Isenring P. Forbush B. Comp. Biochem. Physiol. Part A Mol. Integr. Physiol. 2001; 130: 487-497Crossref PubMed Scopus (59) Google Scholar, 3Payne J.A. Forbush B. Curr. Opin. Cell Biol. 1995; 7: 493-503Crossref PubMed Scopus (108) Google Scholar, 4Russell J.M. Physiol. Rev. 2000; 80: 211-276Crossref PubMed Scopus (734) Google Scholar, 23Lytle C. Forbush B. J. Biol. Chem. 1992; 267: 25438-25443Abstract Full Text PDF PubMed Google Scholar) The CCCs exhibit a common structure, illustrated in Fig. 1A below by a hydropathy plot model of the human (hu) NKCC1. Based on computer-aided analyses from which this model is derived, the CCCs are predicted to have a membrane-associated domain flanked by cytosolic termini. For NKCC1, the membrane localization of the central domain has been confirmed indirectly by the identification of regions that mediate ion transport (24Isenring P. Forbush B. J. Biol. Chem. 1997; 272: 24556-24562Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 25Isenring P. Jacoby S.C. Chang J. Forbush B. J. Gen. Physiol. 1998; 112: 549-558Crossref PubMed Scopus (78) Google Scholar, 26Isenring P. Jacoby S.C. Forbush B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7179-7184Crossref PubMed Scopus (74) Google Scholar) and that behave as transmembrane segments when expressed as fusion proteins in rabbit reticulocyte lysates (27Gerelsaikhan T. Turner R.J. J. Biol. Chem. 2000; 275: 40471-40477Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). For this carrier system, likewise, the intracellular localization of both termini has been confirmed indirectly by the identification of epitope sites that are not accessible unless the lipid bilayer is permeabilized (28Isenring P. Forbush B. Int. Soc. Nephrol. 1997; (abstr.)Google Scholar) and by the identification of phosphoacceptor sites that are involved in transporter regulation (3Payne J.A. Forbush B. Curr. Opin. Cell Biol. 1995; 7: 493-503Crossref PubMed Scopus (108) Google Scholar, 16Darman R.B. Forbush B. J. Biol. Chem. 2002; 277: 37542-37550Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 18Flemmer A.W. Gimenez I. Dowd B.F. Darman R.B. Forbush B. J. Biol. Chem. 2002; 277: 37551-37558Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 19Piechotta K. Lu J. Delpire E. J. Biol. Chem. 2002; 277: 50812-50819Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). Indirect evidence that CCCs are assembled at the cell as homo-oligomeric For of NKCC1, NKCC2, or in membrane to the formation of large the of which is that of the monomeric S. Xu Hebert S.C. J. Am. Soc. Nephrol. 1999; Scholar, Turner R.J. 2000; PubMed Scopus Google Scholar). In with these and have that NKCC1 or the activity of of these in that they a through of the or through with an accessory E. Forbush B. L. Isenring P. Am. J. Physiol. 2003; 284: PubMed Scopus Google Scholar, P. Jacoby S.C. Forbush B. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). the of are at In this study, have the yeast to for of NKCC1 the proximal C as and have a that belongs to the distal C of the have also that the between the regions a large of and that could also our results for the first a self-interacting domain in NKCC1 and by which CCCs regulate and Cell or from the a a a and a and in cells of these also in EGY48 yeast and cells as of reporter or was with the or which to the a the a multiple cloning site a and an resistance which the a the domain of a and the an MCS, a and an which that with the reporter and that are the of a and an which the a glutathione an MCS, and an which the an MCS, and an and which an and a used for the of a that the putative cytosolic C of from residue the transmembrane domain to residue Fig. was from an P. Jacoby S.C. Forbush B. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, S.C. Gagnon E. L. Chang J. Isenring P. Am. J. Physiol. 1999; 277: PubMed Google Scholar) through of the high with an and an The was in which a fusion in yeast and also Fig. The of was also to as an which a fusion in and also Fig. used to and to The are to and sites are in a used to and segments The of these segments and to and to various domains are also of the of the proteins of NKCC1 in in or from first from first from first from first in a In this the of was by segments between a site or that in the of the and a site that in the and are in and and by and the with and The and the of the and the with in and also Fig. For the other segments in through of with an and an in and and also Fig. of the interacting proteins identified in the is from a that the distal C of and also Fig. is in the the domain and was also in as an The a in and also Fig. that are to for the at the of between a site or that in the of and a site that in the and are in and was by the with are called and and also Fig. for the they with an and an or are called and and also Fig. The the in the of an a segment that the cytosolic C to of the was from through of with an and an The was in and also Fig. first transformed with the p8op-lacZ and on the cells transformed with and on The was for of the by below and Fig. and for reporter activation on A for was by cells with a human in and the to of first on and on on X-gal, and activity in yeast to and by to the of the identified cells transformed with a and for of a by below and Fig. of on cells with of which for human has been not to or with other proteins P. R. S. 1993; Google Scholar). and for reporter activation on and that by or used to the sites a for this cells transformed with or and for of a by of on or respectively. the with a or and they for to on or to activity on fusion proteins as and proteins as through E. cells transformed with by in and on glutathione-coupled proteins as from the rabbit reticulocyte The was of of rabbit reticulocyte with amino and or A of the proteins also with a the A or C in and for of the The as was by of proteins on with of for at in a binding and pH by in the to or cell proteins from yeast or on and by or For the was on of the various cell proteins with the by of in with the content of was by and with the and through and the cell used for this is called and of EGY48 yeast with a which the proximal of the cytosolic C terminus. of with a human and of these cells on led to the formation of these on and expressed activity on studies in the yeast that the identified interactions not in the of to the regions of One of the was a that the distal C of was was to the regions in the C of that are for the The that used segments and of these segments for and are illustrated in with of and the which they are is in In Fig. analyses of cells transformed with or that the proteins with an or are at the are in identified as and which no Fig. also that the of individual is in cell or activity cells that transformed with of not to in or The of cells with is in Fig. In and on which cell an and in and they on which an the of that of the and of the used for the mapping are to a to which of for the and to which of for the first of the mapping are in Fig. The various segments that in yeast are by with regions of the C to which they is used to indicate that the segments and is used to indicate that they no in segments by in and for to with in For this they are in the mapping in Fig. is used to indicate that an between and of the mapping studies is in and regions that are for the in the regions that are not in and the regions that may or may not in that the interacting regions are dispersed a in the C terminus. In they to and indicate the residue in and in they to and The also that an between and not unless interacting regions are in of the analyses of the interacting regions identified in this are in Fig. The the amino acid content of interacting and the the of these regions as to the entire C and to various domains as a and The also the of between the interacting regions of NKCC1, NKCC2, and and the of or in the C these is that the interacting regions are the CCCs for the proximal portion of regions are also to residue in and to in and when with other regions in the C terminus. a putative A site is at the of and a site is as and are to play a key in NKCC1 regulation by interacting with the of the carrier (5Xu J.C. Lytle C. Zhu T.T. Payne J.A. Benz E. Forbush B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2201-2205Crossref PubMed Scopus (370) Google Scholar, R.B. Flemmer A. Forbush B. J. Biol. Chem. 2001; 276: 34359-34362Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 16Darman R.B. Forbush B. J. Biol. Chem. 2002; 277: 37542-37550Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, K. M. Turner R.J. Am. J. Physiol. 1999; 277: PubMed Google Scholar) and C as (5Xu J.C. Lytle C. Zhu T.T. Payne J.A. Benz E. Forbush B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2201-2205Crossref PubMed Scopus (370) Google Scholar, 23Lytle C. Forbush B. J. Biol. Chem. 1992; 267: 25438-25443Abstract Full Text PDF PubMed Google Scholar, Am. J. Physiol. 1999; 277: PubMed Google Scholar, J.L. T. Mol. Biol. 1999; PubMed Google Scholar, 2002; PubMed Scopus Google Scholar). sites involved in interactions sites to forkhead-associated binding domains and sites to and domains is that the which is to for a critical residue to a forkhead-associated binding domain site is not to of yeast on studies to the of the yeast or fusion proteins in E. with an or with and they on by in from or predicted and is and on the of the first of the and for this they called and For the as or with of and on with an of in proteins not with the of these studies are In Fig. of the as that are in Based on an the of in is and the of in is In Fig. of that in of and major in and The in probably to and the in to that these at the first of the that used for for the and in and they probably also to the of the indicate that was of a to the formation of and In this system, not a from in a and the first residue of an C. Forbush B. J. Biol. Chem. 1992; 267: 25438-25443Abstract Full Text PDF PubMed Google Scholar); of the high segment from an confirmed that this was the not of the are also in Fig. the of that with as of the of and of For and these are with when with and of that the are for binding for binding and for binding For on the other binding was of the used results that with in and they also that a segment in is for the of the with A or a of not the of proteins that with not In these of with could not by also not or may not in this in system, or the of in the may have been below In this study, have the yeast to of NKCC1. have used the proximal of the cytosolic C as and have identified a that to the distal of the domain studies and confirmed that the between and is for the first that the NKCC1 C possesses self-interacting analyses in which the was used as a mapping to the interacting regions the of contact sites between and In was to from analyses that a of regions and are for the to in these sites at along the C from by studies to the interacting at this of the mapping that the is complex and that probably on of the C terminus. A of the used to sites is that not the by of interacting For involved in the formation of could they in a segment that is also of interacting In of the C from a in NKCC1 could have that in when the carrier is to of the interacting regions are One example that these from the identification of a site that belongs to a forkhead-associated domain that is of an interacting when a residue is from this site in the segment is no to activity in yeast when with the identification of a site in a self-interacting of NKCC1 is an by which CCCs with forkhead-associated binding domains have been to as that phosphorylation-dependent interactions Am. J. Physiol. 1999; 277: PubMed Google Scholar). Based on the of a site in and of a putative site in is to that is the epitope of the in used in this was not to a In the of the C in of NKCC1 has not been in thus a in studies to the NKCC1 C are The of large NKCC1 segments that behave as binding in and that belong to segments of the C to the various conformational that could by this of the are illustrated in Fig. through of the cytosolic C terminus, that the to binding site and the to binding The proposed in this are also on from hydropathy plot analyses of (3Payne J.A. Forbush B. Curr. Opin. Cell Biol. 1995; 7: 493-503Crossref PubMed Scopus (108) Google Scholar, J.C. Lytle C. Zhu T.T. Payne J.A. Benz E. Forbush B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2201-2205Crossref PubMed Scopus (370) Google Scholar, J.A. Xu J.C. M. Lytle Forbush B. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), and they that the interacting segments are both on the cytosolic of the lipid bilayer and by a segment that is also The first of that could by a the formation of between and a result of is predicted that the C a with distal to the central domain. is also predicted that this important in of the C to other For the of between and could the formation of between the regions and the cotransporter to a monomeric or with regulatory The formation of between and is at the of of that could by the cytosolic C terminus. In the of that NKCC1 is as a homo-oligomeric in the in is predicted that of high order could into the of sites that belong to domains of the C terminus. For the C of through a of and for the C of monomeric assembled through of In the with A for and in assembly the that NKCC1 at the cell have not that this was the of evidence the that NKCC1 as a high order of The first of evidence is on a by ion transport systems to in the lipid of cells P. J. J. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, M. Nature. PubMed Scopus Google Scholar, Chang G. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar), and the of is on from which a of CCC proteins has in S. Xu Hebert S.C. J. Am. Soc. Nephrol. 1999; Scholar, Turner R.J. 2000; PubMed Scopus Google Scholar). The common of ion transport systems to as high order has been in by or For high studies have that in lipid as and the of which are by a self-interacting domain in the A. P.J. S. Nature. 1998; PubMed Scopus Google Scholar). For ion transport system, studies have also a for and at the cell assembly of is by binding A. 2000; PubMed Google Scholar). is that other the P. J. J. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar), the P. S. M.A. Am. J. Physiol. 2000; PubMed Google Scholar), are as homo-oligomeric that members of the CCC family are also as homo-oligomeric units has from and Turner Turner R.J. 2000; PubMed Scopus Google Scholar) have that the of NKCC1 could by a when this carrier with of these to no other proteins NKCC1. In the of and Turner Turner R.J. 2000; PubMed Scopus Google Scholar) also that the of a NKCC1 of the cytosolic was to that of the carrier. these the that and are involved in In the of is not to with NKCC1 has a for the monomeric or and and are involved in or Based on the properties to self-interacting that the of and to NKCC1 in the is by the of various enzymes and A.L. Physiol. Rev. 1999; PubMed Scopus Google Scholar), of as homo-oligomeric have been to on the of self-interacting domains A.L. Physiol. Rev. 1999; PubMed Scopus Google Scholar, U. Matthews A. J.L. 2003; PubMed Scopus Google Scholar). for Chang G. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar), is that and NKCC1 to a of at the cell by of the in a The cytosolic C of CCC family members is the 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 (249) Google Scholar, S.C. D.B. Gamba G. J. Physiol. PubMed Scopus Google Scholar). of these in have been to in the cell (1Haas M. Forbush B. Annu. Rev. Physiol. 2000; 62: 515-534Crossref PubMed Scopus (331) Google Scholar, 2Isenring P. Forbush B. Comp. Biochem. Physiol. Part A Mol. Integr. Physiol. 2001; 130: 487-497Crossref PubMed Scopus (59) Google Scholar, 3Payne J.A. Forbush B. Curr. Opin. Cell Biol. 1995; 7: 493-503Crossref PubMed Scopus (108) Google Scholar, 4Russell J.M. Physiol. Rev. 2000; 80: 211-276Crossref PubMed Scopus (734) Google Scholar, S.C. D.B. Gamba G. J. Physiol. PubMed Scopus Google Scholar, J. Delpire E. D.B. Comp. Biochem. Physiol. Part A Mol. Integr. Physiol. 2001; 130: PubMed Scopus Google Scholar). the the of and could also play a in between CCC have been to in the and properties of various ion J. A. T. H. A. M. E. B. N. Acad. Sci. 1999; PubMed Scopus Google Scholar). For the could to the formation of with from of the the this the of individual CCCs on previous In the identification of self-interacting domains in the C of NKCC1 a important in the of cation-Cl- in to the and of CCC proteins and the to at the and of interactions for these transport are to for and to G. and for

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