Nramp2 Transports Ferrous Iron, and is Defective in mk Mice and b Rats • 800

Microcytic anemia (mk) mice and Belgrade (b) rats are inbred rodent strains with well characterized autosomal recessive defects in transmembrane iron transport. Both show abnormalities in intestinal and erythroid iron uptake, but different aspects have been characterized. Enterocytes from mk mice absorb dietary iron poorly, owing to defective apical uptake. Reticulocytes from b rats take up iron through receptor mediated endocytosis of diferric transferrin, but iron fails to leave the endosome, and is exported when the transferrin-transferrin receptor complex is recycled to the cell surface. Using a positional cloning approach, we have recently shown that the same gene, Nramp2, is mutated in both mk mice and b rats (Nature Genetics 1997; 16:383 and Proc. Natl. Acad. Sci. in press). Strikingly, mk and b rats carry exactly the same mutation converting glycine 185 to arginine (G185R). The function of Nramp2 was previously unknown, but the protein has twelve predicted transmembrane domains, characteristic of ion transporters, and others have reported that homologous proteins transport metals in yeast and in Xenopus oocytes. We have now designed a functional assay to test the hypotheses that Nramp2 is an iron transporter, and that the G185R mutation is highly deleterious. Expression constructs encoding wild-type and mutant forms of murine Nramp2 cDNA were transfected into HEK 293T cells, and uptake of ⁵⁵Fe was measured. Wild-type Nramp2 transfectants accumulated approximately 75-fold more radiolabeled iron (Fe²⁺) over 20 minutes than controls. G185R Nramp2 conferred 2-fold more iron uptake than controls, indicating that the mk mutation is highly deleterious for ferrous iron transport. A panel of constructs mutated at other highly conserved residues conferred transport capability intermediate between wild-type and G185R, allowing a structure/function analysis of Nramp2. Biotinylation and immunofluorescence studies indicate that both wild type and G185R Nramp2 localize to the plasma membrane and to transferrin cycle endosomes. Accumulating evidence strongly supports our conclusion that Nramp2 is the major iron transporter in mammals, and that the G185R mutation causes the mk and b phenotypes by disrupting normal iron transport.