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 Co(2+) and Cd(2+) but not Mg(2+) 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.
Picard
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V
Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada.
Govoni
G
G
Jabado
N
N
Gros
P
P
D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D002104:Cadmium / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D027682:Cation Transport Proteins; D002413:Cations, Divalent / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D003035:Cobalt / Q000378:metabolism; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism*; D005452:Fluoresceins / Q000378:metabolism*; D017136:Ion Transport; D007501:Iron / Q000378:metabolism*; D007504:Iron Radioisotopes; D033862:Iron-Binding Proteins; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D000099173:Natural Resistance-associated Macrophage Protein 1; D000099236:Solute Carrier Family 11, Member 2
D002104:Cadmium; D002352:Carrier Proteins; D027682:Cation Transport Proteins; D002413:Cations, Divalent; D003035:Cobalt; D005452:Fluoresceins; D007501:Iron; D007504:Iron Radioisotopes; D033862:Iron-Binding Proteins; D008565:Membrane Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D000099173:Natural Resistance-associated Macrophage Protein 1; D000099236:Solute Carrier Family 11, Member 2; C007740:fluorexon