Publications

• Identification of functional amino acids in the Nramp family by a combination of evolutionary analysis and biophysical studies of metal and proton cotransport in vivo.

  Chaloupka R., Courville P., Veyrier F., Knudsen B., Tompkins T.A., Cellier M.F.

  The natural resistance-associated macrophage protein (Nramp) family is functionally conserved in bacteria and eukarya; Nramp homologues function as proton-dependent membrane transporters of divalent metals. Sequence analyses indicate that five phylogenetic groups comprise the Nramp family, three b ... >> More

  The natural resistance-associated macrophage protein (Nramp) family is functionally conserved in bacteria and eukarya; Nramp homologues function as proton-dependent membrane transporters of divalent metals. Sequence analyses indicate that five phylogenetic groups comprise the Nramp family, three bacterial and two eukaryotic, which are distinct from a more distantly related group of microbial sequences (Nramp outgroup). The Nramp family and outgroup share many conserved residues, suggesting they derived from a common ancestor and raising the possibility that the residues invariant in the Nramp family that correspond to residues which are different but also conserved in the outgroup represent candidate sites of functional divergence of the Nramp family. Four Nramp family-specific residues were identified within transmembrane domains 1, 6, and 11, and replaced by the corresponding invariant outgroup residues in the Escherichia coli Nramp ortholog (the proton-dependent manganese transporter, MntH of group A, EcoliA). The resulting mutants (Asp(34)Gly, Asn(37)Thr, His(211)Tyr, and Asn(401)Gly) were tested for both divalent metal uptake and proton transport; quasi-simultaneous analyses of uptake of metals and protons revealed for the first time protons and metals cotransport by a bacterial Nramp homologue. Additional mutations were studied for comparison (Asp(34)Asn, Asn(37)Asp and Asn(37)Val, Asn(401)Thr, His(211)Ala, His(216)Ala, and His(216)Arg). EcoliA activity was impaired after each of the Nramp/outgroup substitutions, as well as after more conservative replacements, showing that the tested sites are all important for metal uptake and metal-dependent H(+) transport. It is proposed that co-occurrence of these four Nramp-specific transmembrane residues may have contributed to the emergence of this family of metal and proton cotransporters. << Less

  Biochemistry 44:726-733(2005) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Substrate profile and metal-ion selectivity of human divalent metal-ion transporter-1.

  Illing A.C., Shawki A., Cunningham C.L., Mackenzie B.

  Divalent metal-ion transporter-1 (DMT1) is a H(+)-coupled metal-ion transporter that plays essential roles in iron homeostasis. DMT1 exhibits reactivity (based on evoked currents) with a broad range of metal ions; however, direct measurement of transport is lacking for many of its potential substr ... >> More

  Divalent metal-ion transporter-1 (DMT1) is a H(+)-coupled metal-ion transporter that plays essential roles in iron homeostasis. DMT1 exhibits reactivity (based on evoked currents) with a broad range of metal ions; however, direct measurement of transport is lacking for many of its potential substrates. We performed a comprehensive substrate-profile analysis for human DMT1 expressed in RNA-injected Xenopus oocytes by using radiotracer assays and the continuous measurement of transport by fluorescence with the metal-sensitive PhenGreen SK fluorophore. We provide validation for the use of PhenGreen SK fluorescence quenching as a reporter of cellular metal-ion uptake. We determined metal-ion selectivity under fixed conditions using the voltage clamp. Radiotracer and continuous measurement of transport by fluorescence assays revealed that DMT1 mediates the transport of several metal ions that were ranked in selectivity by using the ratio I(max)/K(0.5) (determined from evoked currents at -70 mV): Cd(2+) > Fe(2+) > Co(2+), Mn(2+) ≫ Zn(2+), Ni(2+), VO(2+). DMT1 expression did not stimulate the transport of Cr(2+), Cr(3+), Cu(+), Cu(2+), Fe(3+), Ga(3+), Hg(2+), or VO(+). (55)Fe(2+) transport was competitively inhibited by Co(2+) and Mn(2+). Zn(2+) only weakly inhibited (55)Fe(2+) transport. Our data reveal that DMT1 selects Fe(2+) over its other physiological substrates and provides a basis for predicting the contribution of DMT1 to intestinal, nasal, and pulmonary absorption of metal ions and their cellular uptake in other tissues. Whereas DMT1 is a likely route of entry for the toxic heavy metal cadmium, and may serve the metabolism of cobalt, manganese, and vanadium, we predict that DMT1 should contribute little if at all to the absorption or uptake of zinc. The conclusion in previous reports that copper is a substrate of DMT1 is not supported. << Less

  J. Biol. Chem. 287:30485-30496(2012) [PubMed] [EuropePMC]

  This publication is cited by 6 other entries.

• Unique structural features in an Nramp metal transporter impart substrate-specific proton cotransport and a kinetic bias to favor import.

  Bozzi A.T., Bane L.B., Zimanyi C.M., Gaudet R.

  Natural resistance-associated macrophage protein (Nramp) transporters enable uptake of essential transition metal micronutrients in numerous biological contexts. These proteins are believed to function as secondary transporters that harness the electrochemical energy of proton gradients by "coupli ... >> More

  Natural resistance-associated macrophage protein (Nramp) transporters enable uptake of essential transition metal micronutrients in numerous biological contexts. These proteins are believed to function as secondary transporters that harness the electrochemical energy of proton gradients by "coupling" proton and metal transport. Here we use the <i>Deinococcus radiodurans</i> (Dra) Nramp homologue, for which we have determined crystal structures in multiple conformations, to investigate mechanistic details of metal and proton transport. We untangle the proton-metal coupling behavior of DraNramp into two distinct phenomena: ΔpH stimulation of metal transport rates and metal stimulation of proton transport. Surprisingly, metal type influences substrate stoichiometry, leading to manganese-proton cotransport but cadmium uniport, while proton uniport also occurs. Additionally, a physiological negative membrane potential is required for high-affinity metal uptake. To begin to understand how Nramp's structure imparts these properties, we target a conserved salt-bridge network that forms a proton-transport pathway from the metal-binding site to the cytosol. Mutations to this network diminish voltage and ΔpH dependence of metal transport rates, alter substrate selectivity, perturb or eliminate metal-stimulated proton transport, and erode the directional bias favoring outward-to-inward metal transport under physiological-like conditions. Thus, this unique salt-bridge network may help Nramp-family transporters maximize metal uptake and reduce deleterious back-transport of acquired metals. We provide a new mechanistic model for Nramp proton-metal cotransport and propose that functional advantages may arise from deviations from the traditional model of symport. << Less

  J Gen Physiol 151:1413-1429(2019) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• A novel proton transfer mechanism in the SLC11 family of divalent metal ion transporters.

  Pujol-Gimenez J., Hediger M.A., Gyimesi G.

  In humans, the H⁺-coupled Fe²⁺ transporter DMT1 (SLC11A2) is essential for proper maintenance of iron homeostasis. While X-ray diffraction has recently unveiled the structure of the bacterial homologue ScaDMT as a LeuT-fold transporter, the exact mechanism of H⁺-co ... >> More

  In humans, the H⁺-coupled Fe²⁺ transporter DMT1 (SLC11A2) is essential for proper maintenance of iron homeostasis. While X-ray diffraction has recently unveiled the structure of the bacterial homologue ScaDMT as a LeuT-fold transporter, the exact mechanism of H⁺-cotransport has remained elusive. Here, we used a combination of molecular dynamics simulations, in silico pK _a calculations and site-directed mutagenesis, followed by rigorous functional analysis, to discover two previously uncharacterized functionally relevant residues in hDMT1 that contribute to H⁺-coupling. E193 plays a central role in proton binding, thereby affecting transport properties and electrogenicity, while N472 likely coordinates the metal ion, securing an optimally "closed" state of the protein. Our molecular dynamics simulations provide insight into how H⁺-translocation through E193 is allosterically linked to intracellular gating, establishing a novel transport mechanism distinct from that of other H⁺-coupled transporters. << Less

  Sci Rep 7:6194-6194(2017) [PubMed] [EuropePMC]

• Molecular Mechanism of Nramp-Family Transition Metal Transport.

  Bozzi A.T., Gaudet R.

  The Natural resistance-associated macrophage protein (Nramp) family of transition metal transporters enables uptake and trafficking of essential micronutrients that all organisms must acquire to survive. Two decades after Nramps were identified as proton-driven, voltage-dependent secondary transpo ... >> More

  The Natural resistance-associated macrophage protein (Nramp) family of transition metal transporters enables uptake and trafficking of essential micronutrients that all organisms must acquire to survive. Two decades after Nramps were identified as proton-driven, voltage-dependent secondary transporters, multiple Nramp crystal structures have begun to illustrate the fine details of the transport process and provide a new framework for understanding a wealth of preexisting biochemical data. Here we review the relevant literature pertaining to Nramps' biological roles and especially their conserved molecular mechanism, including our updated understanding of conformational change, metal binding and transport, substrate selectivity, proton transport, proton-metal coupling, and voltage dependence. We ultimately describe how the Nramp family has adapted the LeuT fold common to many secondary transporters to provide selective transition-metal transport with a mechanism that deviates from the canonical model of symport. << Less

  J Mol Biol 433:166991-166991(2021) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Cloning and characterization of a mammalian proton-coupled metal-ion transporter.

  Gunshin H., Mackenzie B., Berger U.V., Gunshin Y., Romero M.F., Boron W.F., Nussberger S., Gollan J.L., Hediger M.A.

  Metal ions are essential cofactors for a wealth of biological processes, including oxidative phosphorylation, gene regulation and free-radical homeostasis. Failure to maintain appropriate levels of metal ions in humans is a feature of hereditary haemochromatosis, disorders of metal-ion deficiency, ... >> More

  Metal ions are essential cofactors for a wealth of biological processes, including oxidative phosphorylation, gene regulation and free-radical homeostasis. Failure to maintain appropriate levels of metal ions in humans is a feature of hereditary haemochromatosis, disorders of metal-ion deficiency, and certain neurodegenerative diseases. Despite their pivotal physiological roles, however, there is no molecular information on how metal ions are actively absorbed by mammalian cells. We have now identified a new metal-ion transporter in the rat, DCT1, which has an unusually broad substrate range that includes Fe2+, Zn2+, Mn2+, Co2+, Cd2+, Cu2+, Ni2+ and Pb2+. DCT1 mediates active transport that is proton-coupled and depends on the cell membrane potential. It is a 561-amino-acid protein with 12 putative membrane-spanning domains and is ubiquitously expressed, most notably in the proximal duodenum. DCT1 is upregulated by dietary iron deficiency, and may represent a key mediator of intestinal iron absorption. DCT1 is a member of the 'natural-resistance-associated macrophage protein' (Nramp) family and thus its properties provide insight into how these proteins confer resistance to pathogens. << Less

  Nature 388:482-488(1997) [PubMed] [EuropePMC]

  This publication is cited by 6 other entries.