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

In mammals, natural-resistance-associated macrophage protein 1 (Nramp1) regulates macrophage activation and is associated with infectious and autoimmune diseases. Nramp2 is associated with anaemia. Both belong to a highly conserved eukaryote/ prokaryote protein family. We used Xenopus oocytes to demonstrate that, like Nramp2, Nramp1 is a bivalent cation (Fe²⁺, Zn²⁺ and Mn²⁺) transporter. Strikingly, however, where Nramp2 is a symporter of H⁺ and metal 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 × 10^-6) and human NRAMP1 (P = 3.87 × 10^-5) showed significantly enhanced uptake of radiolabelled Zn²⁺ compared with water-injected controls. At pH 5.5, Nramp1^G169 (P = 1.34 × 10^-13) and NRAMP1 (P = 1.09 × 10^-6) oocytes showed significant efflux of Zn²⁺. Zn²⁺ transport was abolished when the proton gradient was dissipated using carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Using pre-acidified oocytes, currents of 130 ± 57 nA were evoked by 100 μM Zn²⁺ at pH 7.5, and 139 ± 47 nA by 100 μM Fe²⁺ at pH 7.0, in Nramp1^G169 oocytes; currents of 254 ± 49 nA and 242 ± 26 nA were evoked, respectively, in NRAMP1 oocytes. Steady-state currents evoked by increasing concentrations of Zn²⁺ were saturable, with apparent affinity constants of approx. 614 nM for Nramp1^G169 and approx. 562 nM for NRAMP1 oocytes, and a curvilinear voltage dependence 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 model for metal ion homoeostasis in macrophages. Under normal physiological conditions, Nramp2, localized to early endosomal membranes, delivers extracellularly acquired bivalent cations into the cytosol. Nramp1, localized to late endosomal/lysosomal membranes, delivers bivalent cations from the cytosol into this acidic compartment where they may directly affect antimicrobial activity.