The family of NRAMP metal ion transporters functions in diverse organisms from bacteria to human. NRAMP1 functions in metal transport across the phagosomal membrane of macrophages, and defective NRAMP1 causes sensitivity to several intracellular pathogens. DCT1 (NRAMP2) transport metal ions at the plasma membrane of cells of both the duodenum and in peripheral tissues, and defective DCT1 cause anemia. The driving force for the metal-ion transport is proton gradient (protonmotive force). In DCT1 the stoichiometry between metal ion and proton varied at different conditions due to a mechanistic proton slip. Though the metal ion transport by Smf1p, the yeast homolog of DCT1, is also a protonmotive force, a slippage of sodium ions was observed. The mechanism of the above phenomena could be explained by a combination between transporter and channel mechanisms.

The yeast null mutant smf1⌬ cannot grow on medium containing EGTA. Expression of Smf1p or the mammalian transporter DCT1 (Slc11a2) suppresses the above-mentioned phenotype. Both can also be expressed in Xenopus oocytes, and the uptake activity and their electrophysiological properties can be studied. We used these systems to analyze the properties of mutations in the predicted external loop I of DCT1. The sensitivity of the transporter to amino acid substitutions in this region is manifested by the mutation G119A, which resulted in almost complete inhibition of the metal ion uptake activity and marked changes in the pre-steady-state currents in Xenopus oocytes. The mutation Q126D abolished the uptake and the electrophysiology, but the double mutant D124A͞ Q126D partially restored it and changed the metal ion specificity in favor of Fe 2؉ . The maximal pre-steady-state currents at negatively imposed potentials shifted to a lower pH of Ϸ5. The triple mutant G119A͞D124A͞Q126D, which has no apparent transport activity, exhibited remarkable pre-steady-state currents at pH 7.5. Moreover, Zn 2؉ had a dual effect on this mutant; at pH 7.5 it eliminated the pre-steady state without generating steady-state currents, and at pH 5.5 it induced large pre-steady-state currents. The mutant D124A retained appreciable Fe 2؉ uptake activity but exhibited very little Mn 2؉ uptake at pH 5.5 and was abolished at pH 6.5. The properties of the various mutants suggest that loop I is involved in the metal ion binding and its coupling to the proton-driving force.mutations ͉ uptake ͉ electrophysiology ͉ yeast ͉ oocytes In the last few years it has become apparent that, from bacteria to man, the family of natural resistance-associated macrophage protein (NRAMP) metal ion transporters plays a major role in metal ion homeostasis (1-4). This family is represented in yeast by three genes (SMF1, SMF2, and SMF3) and in mammals by NRAMP1 and NRAMP2 (DCT1). The family members function as general metal ion transporters and can transport not only Mn 2ϩ and Cu 2ϩ (5) but also Fe 2ϩ , Cd 2ϩ , Ni 2ϩ , and Co 2ϩ (6-8). Expression of DCT1 and Smf1p in Xenopus oocytes demonstrated that the driving force for the divalent metal ion transport is H ϩ -dependent (8, 9). In addition, in the expressing oocytes, a large H ϩ slip through DCT1 and Na ϩ slippage through Smf1p was observed (8, 10). The identity of the substrate and proton-binding sites on the transporters are not known, and the mechanism of metal ion transport is obscure. Few conserved amino acids were changed in Smf1p (11) and DCT1 (12, 13), but the results have not yet revealed many aspects of the action mechanism of these transporters. We developed a concerted approach for the study of Smf1p and DCT1. It uses yeast null mutants lacking the SMF metal ion transporters (14), and Xenopus oocytes, where those transporters are absent, as an expression medium for Smf1p and DCT1 in appropriate plasmids (10). The null mutant smf1⌬ is unable to grow in the presence of EGTA (5, 14). Expression of Smf1p or the...

Metal ion transport by DCT1, a member of the natural resistance-associated macrophage protein family, is driven by protons. The stoichiometry of the proton to metal ion is variable, and under optimal transport conditions, more than 10 protons are co-transported with a single metal ion. To understand this phenomenon better, we used site-directed mutagenesis of DCT1 and analyzed the mutants by complementation of yeast suppressor of mitochondria import function-null mutants and electrophysiology with Xenopus oocytes. The mutation F227I resulted in an increase of up to 14-fold in the ratio between metal ions to protons transported. This observation suggests that low metal ion to proton transport of DCT1 resulting from a proton slippage is not a necessity of the transport mechanism in which positively charged protons are driving two positive charges of the metal ion in the same direction. It supports the idea that the proton slippage has a physiological advantage, and the proton slip was positively selected during the evolution of DCT1.Metal ions are vital elements for all living cells. The NRAMP 1 family of metal ion transporters apparently plays a major role in metal ion homeostasis (1-4). Most of the information available about the mechanism of these transporters has resulted from studies of the family members NRAMP2 (DCT1) from mammalian and SMF1 from yeast expressed in Xenopus oocyte (5-9). Xenopus oocytes have a very low metal ion uptake background, which makes them the ideal heterologous expression system for metal ion transporters. In addition to the uptake measurements of the various divalent cations, it is possible to analyze the electrophysiological parameters generated by imposed potentials in the transporter-expressing oocyte as compared with control (10). The studies of DCT1 and Smf1p demonstrated that both of them function as general divalent metal ion transporters and that a proton gradient is the driving force for the metal ion transport (8, 9). However, the transport of protons and metal ions is "loosely coupled" because the process exhibits a variable stoichiometry. At pH 7 and membrane potentials of Ϫ90 to Ϫ30 mV, DCT1 transports one Fe 2ϩ ion with one H ϩ . At high proton concentration (low pH), the number of H ϩ ions transported with one Fe 2ϩ ion increased to 10 (8). Moreover, on changing the membrane potential from ϩ10 to Ϫ80 mV at this low pH, the number of H ϩ ions transported with one Fe 2ϩ ion increased from 3 to ϳ18 (9). In DCT1, this phenomenon was defined as a metal ion-dependent proton slippage (1, 9, 11). In contrast to DCT1, Smf1p showed a metal ion-independent sodium slip through the proton-translocating pathway (9, 12). The mechanism of this phenomenon is not well characterized, and the sites on the transporters that generate it are not known.In this work, we addressed the coupling between proton and metal ion transport, and report on a mutation in DCT1 that exhibits an ϳ14-fold increase in the ratio of metal ion to proton transport. MATERIALS AND METHODSSite-directed Mutagenesis o...

DCT1 (NRAMP2, DMT1, slc11a2) is a member of the NRAMP family and functions as general metal ion transporter in mammals; defective DCT1 causes anemia. The driving force for metal ion transport is protonmotive force, where protons are transported in the same direction as metal ions. The stoichiometry between metal ion and proton varies under different conditions due to mechanistic proton slip. To better understand this phenomenon, we performed site-directed mutagenesis of DCT1 and analyzed the mutants by measurement of metal ion uptake activity and electrophysiology in Xenopus laevis oocytes. A single reciprocal mutation, I144F, between DCT1 and the homologous yeast transporter Smf1p located in putative transmembrane domain 2 abolished the metal ion transport activity of DCT1, significantly increased the slip currents, and generated sodium slip currents. A double mutation adding F227I in transmembrane domain 4 to I144F in transmembrane domain 2 restored the uptake activity of DCT1 and reduced the slip currents. These results demonstrate the importance of these regions in coupling of metal ions and protons as well as the possible proximity of I144 and F227 in the folded structure of DCT1.