Nramp 2 (DCT1/DMT1) Expressed at the Plasma Membrane Transports Iron and Other Divalent Cations into a Calcein-accessible Cytoplasmic Pool

Nramp2, also known as DMT1 and DCT1, is a 12-transmembrane (TM) domain protein responsible for dietary iron uptake in the duodenum and iron acquisition from transferrin in peripheral tissues. Nramp2/DMT1 produces by alternative splicing two isoforms differing at their C terminus (isoforms I and II). The subcellular localization, mechanism of action, and destination of divalent cations transported by the two Nramp2 isoforms are not completely understood. Stable CHO transfectants expressing Nramp2 isoform II modified by addition of a hemaglutinin epitope in the loop defined by the TM7–TM8 interval were generated. Immunofluorescence with permeabilized and intact cells established that Nramp2 isoform II is expressed at the plasma membrane and demonstrated the predicted extracytoplasmic location of the TM7–TM8 loop. Using the fluorescent, metal-sensitive dye calcein, and a combination of membrane-permeant and -impermeant iron chelators, Nramp2 transport was measured and quantitated with respect to kinetic parameters and at steady state. Iron transport at the plasma membrane was time- and pH-dependent, saturable, and proportional to the amount of Nramp2 expression. Iron uptake by Nramp2 at the plasma membrane was into the nonferritin-bound, calcein-accessible so-called “labile iron pool.” Ion selectivity experiments show that Nramp2 isoform II can also transport Co2+ and Cd2+ but not Mg2+ into the calcein-accessible pool. Parallel experiments with transfectants expressing the lysosomal Nramp1 homolog do not show any divalent cation transport activity, establishing major functional differences between Nramp1 and Nramp2. Monitoring the effect of Nramp2 on the calcein-sensisitve labile iron pool allows a simple, rapid, and nonisotopic approach to the functional study of this protein. Nramp2, also known as DMT1 and DCT1, is a 12-transmembrane (TM) domain protein responsible for dietary iron uptake in the duodenum and iron acquisition from transferrin in peripheral tissues. Nramp2/DMT1 produces by alternative splicing two isoforms differing at their C terminus (isoforms I and II). The subcellular localization, mechanism of action, and destination of divalent cations transported by the two Nramp2 isoforms are not completely understood. Stable CHO transfectants expressing Nramp2 isoform II modified by addition of a hemaglutinin epitope in the loop defined by the TM7–TM8 interval were generated. Immunofluorescence with permeabilized and intact cells established that Nramp2 isoform II is expressed at the plasma membrane and demonstrated the predicted extracytoplasmic location of the TM7–TM8 loop. Using the fluorescent, metal-sensitive dye calcein, and a combination of membrane-permeant and -impermeant iron chelators, Nramp2 transport was measured and quantitated with respect to kinetic parameters and at steady state. Iron transport at the plasma membrane was time- and pH-dependent, saturable, and proportional to the amount of Nramp2 expression. Iron uptake by Nramp2 at the plasma membrane was into the nonferritin-bound, calcein-accessible so-called “labile iron pool.” Ion selectivity experiments show that Nramp2 isoform II can also transport Co2+ and Cd2+ but not Mg2+ into the calcein-accessible pool. Parallel experiments with transfectants expressing the lysosomal Nramp1 homolog do not show any divalent cation transport activity, establishing major functional differences between Nramp1 and Nramp2. Monitoring the effect of Nramp2 on the calcein-sensisitve labile iron pool allows a simple, rapid, and nonisotopic approach to the functional study of this protein. transmembrane iron response element Chinese hamster ovary labile iron pool ferrous ammonium sulfate salicyladehyde isocotinoyl hydrazonye 6-desferrioxamine polymerase chain reaction hemagglutinin phosphate-buffered saline bovine serum albumin acetoxymethylester 4-morpholineethanesulfonic acid plasma membrane The Nramp2 gene (naturalresistance-associated macrophageprotein-2), also known as DCT1 (1Gunshin H. Mackenzie B. Berger U.V. Gunshin Y. Romero M.F. Boron W.F. Nussberger S. Gollan J.L. Hediger M.A. Nature. 1997; 388: 482-488Crossref PubMed Scopus (2633) Google Scholar) and DMT1 (2Fleming R.E. Migas M.C. Zhou X. Jiang J. Britton R.S. Brunt E.M. Tomatsu S. Waheed A. Bacon B.R. Sly W.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3143-3148Crossref PubMed Scopus (257) Google Scholar), was first identified in mammals (3Gruenheid S. Cellier M. Vidal S. Gros P. Genomics. 1995; 25: 514-525Crossref PubMed Scopus (252) Google Scholar) and belongs to a large family of integral membrane proteins highly conserved throughout evolution, from bacteria to man (4Cellier M. Prive G. Belouchi A. Kwan Gros P. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google M. J. 1999; PubMed Google H. Gros P. Cellier M.F. PubMed Scopus Google S. J. 1999; PubMed Scopus Google M. J. PubMed Scopus Google in the family into functional to as divalent (4Cellier M. Prive G. Belouchi A. Kwan Gros P. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google M. J. PubMed Scopus Google A. H. Hediger M.A. J. 1999; PubMed Scopus Google S. M. Gros P. J. 1997; PubMed Scopus Google J. Gros P. J. 1999; Google of Nramp2 protein a membrane protein of transmembrane a extracytoplasmic a transport in and transport and conserved in (4Cellier M. Prive G. Belouchi A. Kwan Gros P. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google the membrane of Nramp2 to gene produces by alternative splicing of the two that are by acid and by the of iron response element in the of the PubMed Scopus Google Nramp2 isoform I protein P. M. M. J. Genomics. PubMed Scopus Google Scholar) is expressed at the duodenum is by dietary iron S. P. Gros P. 1999; PubMed Google in and a of iron and with iron at the M.A. W.F. 1997; PubMed Scopus Google M.A. M.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google PubMed Google Proc. PubMed Scopus Google J. PubMed Scopus Google that Nramp2 isoform I is responsible for iron transport from the duodenum into the of plasma membrane for Nramp2 to S. S. S. Gros P. J. 1999; PubMed Scopus Google M.A. PubMed Google Scholar) at the of S. P. Gros P. 1999; PubMed Google of protein in as as CHO and show that Nramp2 is also expressed in a subcellular identified as S. S. S. Gros P. J. 1999; PubMed Scopus Google M.A. PubMed Google Scholar) M. J. PubMed Scopus Google Scholar) that Nramp2, and isoform also in iron acquisition in peripheral as iron the membrane of into the S. S. S. Gros P. J. 1999; PubMed Scopus Google M.A. PubMed Google for Nramp2 isoform II in iron transport at the plasma membrane in to of transport Nramp2 isoform I a of divalent cations as and transport is pH-dependent, and with the of a (1Gunshin H. Mackenzie B. Berger U.V. Gunshin Y. Romero M.F. Boron W.F. Nussberger S. Gollan J.L. Hediger M.A. Nature. 1997; 388: 482-488Crossref PubMed Scopus (2633) Google iron transport was also demonstrated at the membrane of cells S. M. A. M. M. B. P. J. PubMed Scopus Google of the but not Nramp2 in cells in a of uptake M.A. PubMed Google The to iron is by Nramp2 not transport as a and do not between and iron in subcellular in are to the and mechanism of experiments do not the and subcellular of into the and of Nramp2 the of transport and destination of the transported CHO that epitope of Nramp2 were Nramp2 was expressed at the plasma membrane and the epitope between and was to from the that the loop is the metal-sensitive and dye that Nramp2 can as a divalent cation at the plasma membrane on and also show that the iron transported by Nramp2 is to the of CHO the so-called “labile iron S. H. 1997; PubMed Scopus Google of the of the protein family is the of a acid loop by and The of this loop is not conserved throughout for Nramp1 and Nramp2 show of with in this this that this loop and extracytoplasmic M. PubMed Scopus Google with the of a transport in the is conserved at the of was to the predicted of the of the protein (4Cellier M. Prive G. Belouchi A. Kwan Gros P. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google this the of epitope in the loop not protein membrane transport in Nramp2 CHO transfectants with that this loop was in Nramp2. a first of the of the protein on and that this approach for of of mechanism of transport of Nramp2 in (1Gunshin H. Mackenzie B. Berger U.V. Gunshin Y. Romero M.F. Boron W.F. Nussberger S. Gollan J.L. Hediger M.A. Nature. 1997; 388: 482-488Crossref PubMed Scopus (2633) Google Scholar), in in cells M.A. PubMed Google S. M. A. M. M. B. P. J. PubMed Scopus Google Scholar) and by the of are are in and and not the transport of and are that that their iron a to to and proteins in and a in cells the of to at in of transport on the destination of the transported between iron to and the pool of of is that the subcellular of the protein to a is to with to a of divalent and and of and are of of but not to in S. P. J. 1995; PubMed Google Scholar), the with in of the iron transport to in cells the of the so-called labile iron pool S. H. 1997; PubMed Scopus Google S. P. J. Scopus Google Scholar), that also to the of Nramp2 in intact and also on the of iron transported by Nramp2. a combination of and membrane-permeant in were to in a kinetic the effect of Nramp2 on the of the iron pool in CHO of iron in Nramp2 transfectants that was to the amount of Nramp2 protein expressed in a cells a in the of and in CHO cells this Nramp2 transport of is at and is at a in with that measured in for and iron transport (1Gunshin H. Mackenzie B. Berger U.V. Gunshin Y. Romero M.F. Boron W.F. Nussberger S. Gollan J.L. Hediger M.A. Nature. 1997; 388: 482-488Crossref PubMed Scopus (2633) Google A. H. Hediger M.A. J. 1999; PubMed Scopus Google Nramp2 expressed at the plasma membrane is to transport Co2+ and Cd2+ but not The of iron transported at the plasma membrane by iron as Nramp2 in P. 1999; Google to in subcellular membrane as of a pool identified as the S. H. 1997; PubMed Scopus Google from this study show that iron transport into CHO cells by Nramp2 is into the calcein-accessible kinetic and show that of the iron transported by Nramp2 the is into that Nramp2 gene produces by alternative splicing of the two proteins and that are by acid and the of in the of the PubMed Scopus Google The isoform I protein S. P. Gros P. 1999; PubMed Google Scholar) is expressed at the duodenum is by dietary iron and responsible for iron transport from the duodenum into the of is the isoform I of Nramp2 that in transport to the iron transport by Nramp2 (1Gunshin H. Mackenzie B. Berger U.V. Gunshin Y. Romero M.F. Boron W.F. Nussberger S. Gollan J.L. Hediger M.A. Nature. 1997; 388: 482-488Crossref PubMed Scopus (2633) Google in intact cells in CHO and in show that Nramp2 is also expressed in a subcellular identified as S. S. S. Gros P. J. 1999; PubMed Scopus Google M.A. PubMed Google Scholar) M. J. PubMed Scopus Google cells and that the of the protein expressed at that is the isoform II of Nramp2. and with S. S. S. Gros P. J. 1999; PubMed Scopus Google M.A. PubMed Google Scholar) and in in a for Nramp2 in the transport of transferrin iron from into the of peripheral S. H. J. PubMed Google J. Google the to by that the isoform II of the protein in can expressed at the plasma The of transport with the in that Nramp2 isoform II can as a divalent cation at the plasma membrane of The that isoforms I and II can to the in cells and in cells that the membrane for this is not in the C terminus of the between isoforms I and is to that and identified by of the of Nramp2 in this identified as for membrane proteins as transferrin and J. 1999; PubMed Scopus Google J.L. J. PubMed Google PubMed Scopus Google to Nramp2 transport not on the of of Nramp2 of are not is in intact cells and is not and cells in this can the of two allows to from transport and of and kinetic can for transport of divalent cations by this and show that divalent cations transport by Nramp2, is into a pool that is to can to in Nramp2 by of and of the of the in the predicted TM7–TM8 loop not to transport activity, with Nramp2 with a at the C membrane of Nramp2 by in CHO cells expressing proteins with epitope at and for in this Gros P. PubMed Scopus Google to their Nramp2 CHO cells with and expressing of the Nramp1 protein (4Cellier M. Prive G. Belouchi A. Kwan Gros P. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar) do not divalent cations in the The of between Nramp1 and Nramp2 and the that a at the can by Nramp1 in J. Gros P. J. 1999; Google Scholar) in a to that of dietary S. H. J. PubMed Google Scholar), that Nramp1 can transport divalent cations as The of divalent cation transport for Nramp1 CHO cells is to the of the of Nramp1 at the plasma membrane to in the lysosomal of cells S. M. Gros P. J. 1997; PubMed Scopus Google G. S. Gros P. 1999; PubMed Google The with Nramp1 and Nramp2 CHO transfectants that the protein of the two proteins to subcellular can identified in proteins expressed in CHO cells and by this are to for this The Nramp2 gene (naturalresistance-associated macrophageprotein-2), also known as DCT1 (1Gunshin H. Mackenzie B. Berger U.V. Gunshin Y. Romero M.F. Boron W.F. Nussberger S. Gollan J.L. Hediger M.A. Nature. 1997; 388: 482-488Crossref PubMed Scopus (2633) Google Scholar) and DMT1 (2Fleming R.E. Migas M.C. Zhou X. Jiang J. Britton R.S. Brunt E.M. Tomatsu S. Waheed A. Bacon B.R. Sly W.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3143-3148Crossref PubMed Scopus (257) Google Scholar), was first identified in mammals (3Gruenheid S. Cellier M. Vidal S. Gros P. Genomics. 1995; 25: 514-525Crossref PubMed Scopus (252) Google Scholar) and belongs to a large family of integral membrane proteins highly conserved throughout evolution, from bacteria to man (4Cellier M. Prive G. Belouchi A. Kwan Gros P. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google M. J. 1999; PubMed Google H. Gros P. Cellier M.F. PubMed Scopus Google S. J. 1999; PubMed Scopus Google M. J. PubMed Scopus Google in the family into functional to as divalent (4Cellier M. Prive G. Belouchi A. Kwan Gros P. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google M. J. PubMed Scopus Google A. H. Hediger M.A. J. 1999; PubMed Scopus Google S. M. Gros P. J. 1997; PubMed Scopus Google J. Gros P. J. 1999; Google of Nramp2 protein a membrane protein of transmembrane a extracytoplasmic a transport in and transport and conserved in (4Cellier M. Prive G. Belouchi A. Kwan Gros P. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google the membrane of Nramp2 to gene produces by alternative splicing of the two that are by acid and by the of iron response element in the of the PubMed Scopus Google Nramp2 isoform I protein P. M. M. J. Genomics. PubMed Scopus Google Scholar) is expressed at the duodenum is by dietary iron S. P. Gros P. 1999; PubMed Google in and a of iron and with iron at the M.A. W.F. 1997; PubMed Scopus Google M.A. M.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google PubMed Google Proc. PubMed Scopus Google J. PubMed Scopus Google that Nramp2 isoform I is responsible for iron transport from the duodenum into the of plasma membrane for Nramp2 to S. S. S. Gros P. J. 1999; PubMed Scopus Google M.A. PubMed Google Scholar) at the of S. P. Gros P. 1999; PubMed Google of protein in as as CHO and show that Nramp2 is also expressed in a subcellular identified as S. S. S. Gros P. J. 1999; PubMed Scopus Google M.A. PubMed Google Scholar) M. J. PubMed Scopus Google Scholar) that Nramp2, and isoform also in iron acquisition in peripheral as iron the membrane of into the S. S. S. Gros P. J. 1999; PubMed Scopus Google M.A. PubMed Google for Nramp2 isoform II in iron transport at the plasma membrane in to The of transport Nramp2 isoform I a of divalent cations as and transport is pH-dependent, and with the of a (1Gunshin H. Mackenzie B. Berger U.V. Gunshin Y. Romero M.F. Boron W.F. Nussberger S. Gollan J.L. Hediger M.A. Nature. 1997; 388: 482-488Crossref PubMed Scopus (2633) Google iron transport was also demonstrated at the membrane of cells S. M. A. M. M. B. P. J. PubMed Scopus Google of the but not Nramp2 in cells in a of uptake M.A. PubMed Google The to iron is by Nramp2 not transport as a and do not between and iron in subcellular in are to the and mechanism of experiments do not the and subcellular of into the and of Nramp2 the of transport and destination of the transported CHO that epitope of Nramp2 were Nramp2 was expressed at the plasma membrane and the epitope between and was to from the that the loop is the metal-sensitive and dye that Nramp2 can as a divalent cation at the plasma membrane on and also show that the iron transported by Nramp2 is to the of CHO the so-called “labile iron S. H. 1997; PubMed Scopus Google of the of the protein family is the of a acid loop by and The of this loop is not conserved throughout for Nramp1 and Nramp2 show of with in this this that this loop and extracytoplasmic M. PubMed Scopus Google with the of a transport in the is conserved at the of was to the predicted of the of the protein (4Cellier M. Prive G. Belouchi A. Kwan Gros P. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google this the of epitope in the loop not protein membrane transport in Nramp2 CHO transfectants with that this loop was in Nramp2. a first of the of the protein on and that this approach for of of mechanism of transport of Nramp2 in (1Gunshin H. Mackenzie B. Berger U.V. Gunshin Y. Romero M.F. Boron W.F. Nussberger S. Gollan J.L. Hediger M.A. Nature. 1997; 388: 482-488Crossref PubMed Scopus (2633) Google Scholar), in in cells M.A. PubMed Google S. M. A. M. M. B. P. J. PubMed Scopus Google Scholar) and by the of are are in and and not the transport of and are that that their iron a to to and proteins in and a in cells the of to at in of transport on the destination of the transported between iron to and the pool of of is that the subcellular of the protein to a is to with to a of divalent and and of and are of of but not to in S. P. J. 1995; PubMed Google Scholar), the with in of the iron transport to in cells the of the so-called labile iron pool S. H. 1997; PubMed Scopus Google S. P. J. Scopus Google Scholar), that also to the of Nramp2 in intact and also on the of iron transported by Nramp2. a combination of and membrane-permeant in were to in a kinetic the effect of Nramp2 on the of the iron pool in CHO of iron in Nramp2 transfectants that was to the amount of Nramp2 protein expressed in a cells a in the of and in CHO cells this Nramp2 transport of is at and is at a in with that measured in for and iron transport (1Gunshin H. Mackenzie B. Berger U.V. Gunshin Y. Romero M.F. Boron W.F. Nussberger S. Gollan J.L. Hediger M.A. Nature. 1997; 388: 482-488Crossref PubMed Scopus (2633) Google A. H. Hediger M.A. J. 1999; PubMed Scopus Google Nramp2 expressed at the plasma membrane is to transport Co2+ and Cd2+ but not The of iron transported at the plasma membrane by iron as Nramp2 in P. 1999; Google to in subcellular membrane as of a pool identified as the S. H. 1997; PubMed Scopus Google from this study show that iron transport into CHO cells by Nramp2 is into the calcein-accessible kinetic and show that of the iron transported by Nramp2 the is into that Nramp2 gene produces by alternative splicing of the two proteins and that are by acid and the of in the of the PubMed Scopus Google The isoform I protein S. P. Gros P. 1999; PubMed Google Scholar) is expressed at the duodenum is by dietary iron and responsible for iron transport from the duodenum into the of is the isoform I of Nramp2 that in transport to the iron transport by Nramp2 (1Gunshin H. Mackenzie B. Berger U.V. Gunshin Y. Romero M.F. Boron W.F. Nussberger S. Gollan J.L. Hediger M.A. Nature. 1997; 388: 482-488Crossref PubMed Scopus (2633) Google in intact cells in CHO and in show that Nramp2 is also expressed in a subcellular identified as S. S. S. Gros P. J. 1999; PubMed Scopus Google M.A. PubMed Google Scholar) M. J. PubMed Scopus Google cells and that the of the protein expressed at that is the isoform II of Nramp2. and with S. S. S. Gros P. J. 1999; PubMed Scopus Google M.A. PubMed Google Scholar) and in in a for Nramp2 in the transport of transferrin iron from into the of peripheral S. H. J. PubMed Google J. Google the to by that the isoform II of the protein in can expressed at the plasma The of transport with the in that Nramp2 isoform II can as a divalent cation at the plasma membrane of The that isoforms I and II can to the in cells and in cells that the membrane for this is not in the C terminus of the between isoforms I and is to that and identified by of the of Nramp2 in this identified as for membrane proteins as transferrin and J. 1999; PubMed Scopus Google J.L. J. PubMed Google PubMed Scopus Google to Nramp2 transport not on the of of Nramp2 of are not is in intact cells and is not and cells in this can the of two allows to from transport and of and kinetic can for transport of divalent cations by this and show that divalent cations transport by Nramp2, is into a pool that is to can to in Nramp2 by of and of the of the in the predicted TM7–TM8 loop not to transport activity, with Nramp2 with a at the C membrane of Nramp2 by in CHO cells expressing proteins with epitope at and for in this Gros P. PubMed Scopus Google to their Nramp2 CHO cells with and expressing of the Nramp1 protein (4Cellier M. Prive G. Belouchi A. Kwan Gros P. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar) do not divalent cations in the The of between Nramp1 and Nramp2 and the that a at the can by Nramp1 in J. Gros P. J. 1999; Google Scholar) in a to that of dietary S. H. J. PubMed Google Scholar), that Nramp1 can transport divalent cations as The of divalent cation transport for Nramp1 CHO cells is to the of the of Nramp1 at the plasma membrane to in the lysosomal of cells S. M. Gros P. J. 1997; PubMed Scopus Google G. S. Gros P. 1999; PubMed Google The with Nramp1 and Nramp2 CHO transfectants that the protein of the two proteins to subcellular can identified in proteins expressed in CHO cells and by this are to for this of the of the protein family is the of a acid loop by and The of this loop is not conserved throughout for Nramp1 and Nramp2 show of with in this this that this loop and extracytoplasmic M. PubMed Scopus Google with the of a transport in the is conserved at the of was to the predicted of the of the protein (4Cellier M. Prive G. Belouchi A. Kwan Gros P. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google this the of epitope in the loop not protein membrane transport in Nramp2 CHO transfectants with that this loop was in Nramp2. a first of the of the protein on and that this approach for of of Nramp2. The mechanism of transport of Nramp2 in (1Gunshin H. Mackenzie B. Berger U.V. Gunshin Y. Romero M.F. Boron W.F. Nussberger S. Gollan J.L. Hediger M.A. Nature. 1997; 388: 482-488Crossref PubMed Scopus (2633) Google Scholar), in in cells M.A. PubMed Google S. M. A. M. M. B. P. J. PubMed Scopus Google Scholar) and by the of are are in and and not the transport of and are that that their iron a to to and proteins in and a in cells the of to at in of transport on the destination of the transported between iron to and the pool of of is that the subcellular of the protein to a is to with to a of divalent and and of and are of of but not to in S. P. J. 1995; PubMed Google Scholar), the with in of the iron transport to in cells the of the so-called labile iron pool S. H. 1997; PubMed Scopus Google S. P. J. Scopus Google Scholar), that also to the of Nramp2 in intact and also on the of iron transported by Nramp2. a combination of and membrane-permeant in were to in a kinetic the effect of Nramp2 on the of the iron pool in CHO of iron in Nramp2 transfectants that was to the amount of Nramp2 protein expressed in a cells a in the of and in CHO cells this Nramp2 transport of is at and is at a in with that measured in for and iron transport (1Gunshin H. Mackenzie B. Berger U.V. Gunshin Y. Romero M.F. Boron W.F. Nussberger S. Gollan J.L. Hediger M.A. Nature. 1997; 388: 482-488Crossref PubMed Scopus (2633) Google A. H. Hediger M.A. J. 1999; PubMed Scopus Google Nramp2 expressed at the plasma membrane is to transport Co2+ and Cd2+ but not The of iron transported at the plasma membrane by iron as Nramp2 in P. 1999; Google to in subcellular membrane as of a pool identified as the S. H. 1997; PubMed Scopus Google from this study show that iron transport into CHO cells by Nramp2 is into the calcein-accessible kinetic and show that of the iron transported by Nramp2 the is into that pool. The Nramp2 gene produces by alternative splicing of the two proteins and that are by acid and the of in the of the PubMed Scopus Google The isoform I protein S. P. Gros P. 1999; PubMed Google Scholar) is expressed at the duodenum is by dietary iron and responsible for iron transport from the duodenum into the of is the isoform I of Nramp2 that in transport to the iron transport by Nramp2 (1Gunshin H. Mackenzie B. Berger U.V. Gunshin Y. Romero M.F. Boron W.F. Nussberger S. Gollan J.L. Hediger M.A. Nature. 1997; 388: 482-488Crossref PubMed Scopus (2633) Google in intact cells in CHO and in show that Nramp2 is also expressed in a subcellular identified as S. S. S. Gros P. J. 1999; PubMed Scopus Google M.A. PubMed Google Scholar) M. J. PubMed Scopus Google cells and that the of the protein expressed at that is the isoform II of Nramp2. and with S. S. S. Gros P. J. 1999; PubMed Scopus Google M.A. PubMed Google Scholar) and in in a for Nramp2 in the transport of transferrin iron from into the of peripheral S. H. J. PubMed Google J. Google the to by that the isoform II of the protein in can expressed at the plasma The of transport with the in that Nramp2 isoform II can as a divalent cation at the plasma membrane of The that isoforms I and II can to the in cells and in cells that the membrane for this is not in the C terminus of the between isoforms I and is to that and identified by of the of Nramp2 in this identified as for membrane proteins as transferrin and J. 1999; PubMed Scopus Google J.L. J. PubMed Google PubMed Scopus Google The to Nramp2 transport not on the of of Nramp2 of are not is in intact cells and is not and cells in this can the of two allows to from transport and of and kinetic can for transport of divalent cations by this and show that divalent cations transport by Nramp2, is into a pool that is to can to in Nramp2 by of and of the of the in the predicted TM7–TM8 loop not to transport activity, with Nramp2 with a at the C membrane of Nramp2 by in CHO cells expressing proteins with epitope at and for in this Gros P. PubMed Scopus Google to their Nramp2 CHO cells with and expressing of the Nramp1 protein (4Cellier M. Prive G. Belouchi A. Kwan Gros P. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar) do not divalent cations in the The of between Nramp1 and Nramp2 and the that a at the can by Nramp1 in J. Gros P. J. 1999; Google Scholar) in a to that of dietary S. H. J. PubMed Google Scholar), that Nramp1 can transport divalent cations as The of divalent cation transport for Nramp1 CHO cells is to the of the of Nramp1 at the plasma membrane to in the lysosomal of cells S. M. Gros P. J. 1997; PubMed Scopus Google G. S. Gros P. 1999; PubMed Google The with Nramp1 and Nramp2 CHO transfectants that the protein of the two proteins to subcellular can identified in proteins expressed in CHO cells and by this are to for this are to for this are to for this are to for this

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