Natural-resistance-associated macrophage protein 1 is an H+/bivalent cation antiporter

In mammals, natural-resistance-associated macrophage protein 1 (Nramp1) reg ulates macrophage activation and is associated with infectious and autoimmu ne diseases. Nramp2 is associated with anaemia. Both belong to a highly con served eukaryote/prokaryote protein family. We used Xenopus oocytes to demo nstrate that like Nramp2, Nramp1 is a bivalent cation (Fe2+, Zn2+ and Mn2+) transporter. Strikingly, however, where Nramp2 is a symporter of H+ and me tal ions, Nramp1 is a highly pH-dependent antiporter that fluxes metal ions in either direction against a proton gradient. At pH 9.0, oocytes injected with cRNA from wild-type murine Nramp1 with a glycine residue at position 169 (Nramp1(G169); P = 3.22 x 10(-) (6)) and human NRAMP1 (P = 3.87 x 10(-5 )) showed significantly enhanced uptake of radiolabelled Zn2+ compared with water-injected controls. At pH 5.5, Nramp1G(169) (P = 1.34 x 10(-13)) and NRAMP1 (P = 1.09 x 10 (6)) oocytes showed significant efflux of Zn2+. Zn2transport was abolished when the proton gradient was dissipated using carbo nyl cyanide p-trifluoromethoxyphenylhydrazone. Using pre-acidified oocytes, currents of 130 +/- 57 nA were evoked by 100 muM Zn2+ at pH 7.5, and 139 /- 47 nA by 100 muM Fe2+ at pH 7.0, in Nramp1(G169) oocytes; currents of 25 4 +/- 49 nA and 242 +/- 26 nA were evoked, respectively, in NRAMP1 oocytes. Steady-state currents evoked by increasing concentrations of Zn2+ were sat urable, with apparent affinity constants of approx. 614 nM for Nramp1(G169) and approx. 562 nM for NRAMP1 oocytes, and a curvilinear voltage dependenc e of transporter activity (i.e. the data points approximate to a curve that approaches a linear asymptote). In the present study we propose a new mode l for metal ion homoeostasis in macrophages. Under normal physiological con ditions, Nramp2, localized to early endosomal membranes, delivers extracell ularly acquired bivalent cations into the cytosol. Nramp1, localized to lat e endosomal/lysosomal membranes, delivers bivalent cations from the cytosol into the acidic compartment where they may directly affect antimicrobial a ctivity.