Iron, manganese, and cobalt transport by Nramp1 (Slc11a1) and Nramp2 (Slc11a2) expressed at the plasma membrane

Mutations in the Nramp1 gene (Slc11a1) cause susceptibility to infection by intracellular pathogens. The Nramp1 protein is expressed at the phagosomal membrane of macrophages and neutrophils and is a paralog of the Nramp2 (Slc11a2) iron transporter. The Nramp1 transport mechanism at the phagosomal membrane has remained controversial. An Nramp1 protein modified by insertion of a hemagglutinin epitope into the predicted TM7/8 loop was expressed at the plasma membrane of Chinese hamster ovary cells as demonstrated by immunofluorescence and surface biotinylation. Experiments in Nramp1 HA transfectants using the metal-sensitive fluorophors calcein and Fura2 showed that Nramp1 HA can mediate Fe2+, Mn2+, and Co2+ uptake. Similar results were obtained in transport studies using radioisotopic Fe-55(2+) and Mn-54(2+). Nramp1HA transport was dependent on time, temperature, and acidic pH, occurring down the proton gradient. These experiments suggest that Nramp1HA may be a more efficient transporter of Mn2+ compared to Fe2+ and a more efficient Mn2+ transporter than Nramp2HA. The membrane topology and transport properties of Nramp1HA and Nramp2HA were indistinguishable, suggesting that Nramp1 divalent-metal transport at the phagosomal membrane is mechanistically similar to that of Nramp2 at the membrane of acidified endosomes. These results clarify the mechanism by which Nramp1 contributes to phagocyte defenses against infections.