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. 2001 Mar 15;354(Pt 3):511–519. doi: 10.1042/0264-6021:3540511

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

T Goswami 1, A Bhattacharjee 1, P Babal 1, S Searle 1, E Moore 1, M Li 1, J M Blackwell 1
PMCID: PMC1221682  PMID: 11237855

Abstract

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=3.22x10(-6)) and human NRAMP1 (P=3.87x10(-5)) showed significantly enhanced uptake of radiolabelled Zn2+ compared with water-injected controls. At pH 5.5, Nramp1(G169) (P=1.34x10(-13)) and NRAMP1 (P=1.09x10(-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 microM Zn2+ at pH 7.5, and 139+/-47 nA by 100 microM 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.

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Selected References

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  1. Aballay A., Sarrouf M. N., Colombo M. I., Stahl P. D., Mayorga L. S. Zn2+ depletion blocks endosome fusion. Biochem J. 1995 Dec 15;312(Pt 3):919–923. doi: 10.1042/bj3120919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Abel L., Sánchez F. O., Oberti J., Thuc N. V., Hoa L. V., Lap V. D., Skamene E., Lagrange P. H., Schurr E. Susceptibility to leprosy is linked to the human NRAMP1 gene. J Infect Dis. 1998 Jan;177(1):133–145. doi: 10.1086/513830. [DOI] [PubMed] [Google Scholar]
  3. Atkinson P. G., Barton C. H. Ectopic expression of Nramp1 in COS-1 cells modulates iron accumulation. FEBS Lett. 1998 Mar 27;425(2):239–242. doi: 10.1016/s0014-5793(98)00236-1. [DOI] [PubMed] [Google Scholar]
  4. Barton C. H., Whitehead S. H., Blackwell J. M. Nramp transfection transfers Ity/Lsh/Bcg-related pleiotropic effects on macrophage activation: influence on oxidative burst and nitric oxide pathways. Mol Med. 1995 Mar;1(3):267–279. [PMC free article] [PubMed] [Google Scholar]
  5. Bellamy R., Ruwende C., Corrah T., McAdam K. P., Whittle H. C., Hill A. V. Variations in the NRAMP1 gene and susceptibility to tuberculosis in West Africans. N Engl J Med. 1998 Mar 5;338(10):640–644. doi: 10.1056/NEJM199803053381002. [DOI] [PubMed] [Google Scholar]
  6. Blackwell J. M., Searle S. Genetic regulation of macrophage activation: understanding the function of Nramp1 (=Ity/Lsh/Bcg). Immunol Lett. 1999 Jan;65(1-2):73–80. doi: 10.1016/s0165-2478(98)00127-8. [DOI] [PubMed] [Google Scholar]
  7. Blackwell J. M., Searle S., Goswami T., Miller E. N. Understanding the multiple functions of Nramp1. Microbes Infect. 2000 Mar;2(3):317–321. doi: 10.1016/s1286-4579(00)00295-1. [DOI] [PubMed] [Google Scholar]
  8. Brown D. H., Lafuse W. P., Zwilling B. S. Stabilized expression of mRNA is associated with mycobacterial resistance controlled by Nramp1. Infect Immun. 1997 Feb;65(2):597–603. doi: 10.1128/iai.65.2.597-603.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Canonne-Hergaux F., Gruenheid S., Ponka P., Gros P. Cellular and subcellular localization of the Nramp2 iron transporter in the intestinal brush border and regulation by dietary iron. Blood. 1999 Jun 15;93(12):4406–4417. [PubMed] [Google Scholar]
  10. Cellier M., Belouchi A., Gros P. Resistance to intracellular infections: comparative genomic analysis of Nramp. Trends Genet. 1996 Jun;12(6):201–204. doi: 10.1016/0168-9525(96)30042-5. [DOI] [PubMed] [Google Scholar]
  11. Fleming M. D., Romano M. A., Su M. A., Garrick L. M., Garrick M. D., Andrews N. C. Nramp2 is mutated in the anemic Belgrade (b) rat: evidence of a role for Nramp2 in endosomal iron transport. Proc Natl Acad Sci U S A. 1998 Feb 3;95(3):1148–1153. doi: 10.1073/pnas.95.3.1148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fleming M. D., Trenor C. C., 3rd, Su M. A., Foernzler D., Beier D. R., Dietrich W. F., Andrews N. C. Microcytic anaemia mice have a mutation in Nramp2, a candidate iron transporter gene. Nat Genet. 1997 Aug;16(4):383–386. doi: 10.1038/ng0897-383. [DOI] [PubMed] [Google Scholar]
  13. Fritz P., Saal J. G., Wicherek C., König A., Laschner W., Rautenstrauch H. Quantitative photometrical assessment of iron deposits in synovial membranes in different joint diseases. Rheumatol Int. 1996;15(5):211–216. doi: 10.1007/BF00290523. [DOI] [PubMed] [Google Scholar]
  14. Garner B., Roberg K., Brunk U. T. Endogenous ferritin protects cells with iron-laden lysosomes against oxidative stress. Free Radic Res. 1998 Aug;29(2):103–114. doi: 10.1080/10715769800300121. [DOI] [PubMed] [Google Scholar]
  15. Gerlach M., Ben-Shachar D., Riederer P., Youdim M. B. Altered brain metabolism of iron as a cause of neurodegenerative diseases? J Neurochem. 1994 Sep;63(3):793–807. doi: 10.1046/j.1471-4159.1994.63030793.x. [DOI] [PubMed] [Google Scholar]
  16. Gomes M. S., Appelberg R. Evidence for a link between iron metabolism and Nramp1 gene function in innate resistance against Mycobacterium avium. Immunology. 1998 Oct;95(2):165–168. doi: 10.1046/j.1365-2567.1998.00630.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Goswitz V. C., Brooker R. J. Structural features of the uniporter/symporter/antiporter superfamily. Protein Sci. 1995 Mar;4(3):534–537. doi: 10.1002/pro.5560040319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Griffith J. K., Baker M. E., Rouch D. A., Page M. G., Skurray R. A., Paulsen I. T., Chater K. F., Baldwin S. A., Henderson P. J. Membrane transport proteins: implications of sequence comparisons. Curr Opin Cell Biol. 1992 Aug;4(4):684–695. doi: 10.1016/0955-0674(92)90090-y. [DOI] [PubMed] [Google Scholar]
  19. Gruenheid S., Canonne-Hergaux F., Gauthier S., Hackam D. J., Grinstein S., Gros P. The iron transport protein NRAMP2 is an integral membrane glycoprotein that colocalizes with transferrin in recycling endosomes. J Exp Med. 1999 Mar 1;189(5):831–841. doi: 10.1084/jem.189.5.831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gruenheid S., Pinner E., Desjardins M., Gros P. Natural resistance to infection with intracellular pathogens: the Nramp1 protein is recruited to the membrane of the phagosome. J Exp Med. 1997 Feb 17;185(4):717–730. doi: 10.1084/jem.185.4.717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gunshin H., Mackenzie B., Berger U. V., Gunshin Y., Romero M. F., Boron W. F., Nussberger S., Gollan J. L., Hediger M. A. Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature. 1997 Jul 31;388(6641):482–488. doi: 10.1038/41343. [DOI] [PubMed] [Google Scholar]
  22. Hackam D. J., Rotstein O. D., Zhang W., Gruenheid S., Gros P., Grinstein S. Host resistance to intracellular infection: mutation of natural resistance-associated macrophage protein 1 (Nramp1) impairs phagosomal acidification. J Exp Med. 1998 Jul 20;188(2):351–364. doi: 10.1084/jem.188.2.351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hirsch E. C., Faucheux B. A. Iron metabolism and Parkinson's disease. Mov Disord. 1998;13 (Suppl 1):39–45. [PubMed] [Google Scholar]
  24. Hofmeister A., Neibergs H. L., Pokorny R. M., Galandiuk S. The natural resistance-associated macrophage protein gene is associated with Crohn's disease. Surgery. 1997 Aug;122(2):173–179. doi: 10.1016/s0039-6060(97)90006-4. [DOI] [PubMed] [Google Scholar]
  25. Hughes A. L. Protein phylogenies provide evidence of a radical discontinuity between arthropod and vertebrate immune systems. Immunogenetics. 1998 Mar;47(4):283–296. doi: 10.1007/s002510050360. [DOI] [PubMed] [Google Scholar]
  26. Jabado N., Jankowski A., Dougaparsad S., Picard V., Grinstein S., Gros P. Natural resistance to intracellular infections: natural resistance-associated macrophage protein 1 (Nramp1) functions as a pH-dependent manganese transporter at the phagosomal membrane. J Exp Med. 2000 Nov 6;192(9):1237–1248. doi: 10.1084/jem.192.9.1237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Jellinger K. A., Kienzl E., Rumpelmaier G., Paulus W., Riederer P., Stachelberger H., Youdim M. B., Ben-Shachar D. Iron and ferritin in substantia nigra in Parkinson's disease. Adv Neurol. 1993;60:267–272. [PubMed] [Google Scholar]
  28. Krantz S. B. Pathogenesis and treatment of the anemia of chronic disease. Am J Med Sci. 1994 May;307(5):353–359. doi: 10.1097/00000441-199405000-00009. [DOI] [PubMed] [Google Scholar]
  29. Kuhn D. E., Baker B. D., Lafuse W. P., Zwilling B. S. Differential iron transport into phagosomes isolated from the RAW264.7 macrophage cell lines transfected with Nramp1Gly169 or Nramp1Asp169. J Leukoc Biol. 1999 Jul;66(1):113–119. doi: 10.1002/jlb.66.1.113. [DOI] [PubMed] [Google Scholar]
  30. Lang T., Prina E., Sibthorpe D., Blackwell J. M. Nramp1 transfection transfers Ity/Lsh/Bcg-related pleiotropic effects on macrophage activation: influence on antigen processing and presentation. Infect Immun. 1997 Feb;65(2):380–386. doi: 10.1128/iai.65.2.380-386.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lee P. L., Gelbart T., West C., Halloran C., Beutler E. The human Nramp2 gene: characterization of the gene structure, alternative splicing, promoter region and polymorphisms. Blood Cells Mol Dis. 1998 Jun;24(2):199–215. doi: 10.1006/bcmd.1998.0186. [DOI] [PubMed] [Google Scholar]
  32. Liu X. F., Culotta V. C. Mutational analysis of Saccharomyces cerevisiae Smf1p, a member of the Nramp family of metal transporters. J Mol Biol. 1999 Jun 18;289(4):885–891. doi: 10.1006/jmbi.1999.2815. [DOI] [PubMed] [Google Scholar]
  33. Liu X. F., Culotta V. C. Post-translation control of Nramp metal transport in yeast. Role of metal ions and the BSD2 gene. J Biol Chem. 1999 Feb 19;274(8):4863–4868. doi: 10.1074/jbc.274.8.4863. [DOI] [PubMed] [Google Scholar]
  34. Marger M. D., Saier M. H., Jr A major superfamily of transmembrane facilitators that catalyse uniport, symport and antiport. Trends Biochem Sci. 1993 Jan;18(1):13–20. doi: 10.1016/0968-0004(93)90081-w. [DOI] [PubMed] [Google Scholar]
  35. Multhaup G. Amyloid precursor protein, copper and Alzheimer's disease. Biomed Pharmacother. 1997;51(3):105–111. doi: 10.1016/s0753-3322(97)86907-7. [DOI] [PubMed] [Google Scholar]
  36. Nelson N. Energizing porters by proton-motive force. J Exp Biol. 1994 Nov;196:7–13. doi: 10.1242/jeb.196.1.7. [DOI] [PubMed] [Google Scholar]
  37. Nelson N. Metal ion transporters and homeostasis. EMBO J. 1999 Aug 16;18(16):4361–4371. doi: 10.1093/emboj/18.16.4361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Orgad S., Nelson H., Segal D., Nelson N. Metal ions suppress the abnormal taste behavior of the Drosophila mutant malvolio. J Exp Biol. 1998 Jan;201(Pt 1):115–120. doi: 10.1242/jeb.201.1.115. [DOI] [PubMed] [Google Scholar]
  39. Picard V., Govoni G., Jabado N., Gros P. Nramp 2 (DCT1/DMT1) expressed at the plasma membrane transports iron and other divalent cations into a calcein-accessible cytoplasmic pool. J Biol Chem. 2000 Nov 17;275(46):35738–35745. doi: 10.1074/jbc.M005387200. [DOI] [PubMed] [Google Scholar]
  40. Pinner E., Gruenheid S., Raymond M., Gros P. Functional complementation of the yeast divalent cation transporter family SMF by NRAMP2, a member of the mammalian natural resistance-associated macrophage protein family. J Biol Chem. 1997 Nov 14;272(46):28933–28938. doi: 10.1074/jbc.272.46.28933. [DOI] [PubMed] [Google Scholar]
  41. Rodrigues V., Cheah P. Y., Ray K., Chia W. malvolio, the Drosophila homologue of mouse NRAMP-1 (Bcg), is expressed in macrophages and in the nervous system and is required for normal taste behaviour. EMBO J. 1995 Jul 3;14(13):3007–3020. doi: 10.1002/j.1460-2075.1995.tb07303.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sanjeevi C. B., Miller E. N., Dabadghao P., Rumba I., Shtauvere A., Denisova A., Clayton D., Blackwell J. M. Polymorphism at NRAMP1 and D2S1471 loci associated with juvenile rheumatoid arthritis. Arthritis Rheum. 2000 Jun;43(6):1397–1404. doi: 10.1002/1529-0131(200006)43:6<1397::AID-ANR25>3.0.CO;2-6. [DOI] [PubMed] [Google Scholar]
  43. Searle S., Bright N. A., Roach T. I., Atkinson P. G., Barton C. H., Meloen R. H., Blackwell J. M. Localisation of Nramp1 in macrophages: modulation with activation and infection. J Cell Sci. 1998 Oct;111(Pt 19):2855–2866. doi: 10.1242/jcs.111.19.2855. [DOI] [PubMed] [Google Scholar]
  44. Shaw M. A., Clayton D., Atkinson S. E., Williams H., Miller N., Sibthorpe D., Blackwell J. M. Linkage of rheumatoid arthritis to the candidate gene NRAMP1 on 2q35. J Med Genet. 1996 Aug;33(8):672–677. doi: 10.1136/jmg.33.8.672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Supek F., Supekova L., Nelson H., Nelson N. A yeast manganese transporter related to the macrophage protein involved in conferring resistance to mycobacteria. Proc Natl Acad Sci U S A. 1996 May 14;93(10):5105–5110. doi: 10.1073/pnas.93.10.5105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Varela M. F., Griffith J. K. Nucleotide and deduced protein sequences of the class D tetracycline resistance determinant: relationship to other antimicrobial transport proteins. Antimicrob Agents Chemother. 1993 Jun;37(6):1253–1258. doi: 10.1128/aac.37.6.1253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Vidal S. M., Malo D., Vogan K., Skamene E., Gros P. Natural resistance to infection with intracellular parasites: isolation of a candidate for Bcg. Cell. 1993 May 7;73(3):469–485. doi: 10.1016/0092-8674(93)90135-d. [DOI] [PubMed] [Google Scholar]
  48. Wall D. A., Patel S. Isolation of plasma membrane complexes from Xenopus oocytes. J Membr Biol. 1989 Feb;107(2):189–201. doi: 10.1007/BF01871724. [DOI] [PubMed] [Google Scholar]
  49. Yip R., Dallman P. R. The roles of inflammation and iron deficiency as causes of anemia. Am J Clin Nutr. 1988 Nov;48(5):1295–1300. doi: 10.1093/ajcn/48.5.1295. [DOI] [PubMed] [Google Scholar]
  50. Yuan X. M., Anders W. L., Olsson A. G., Brunk U. T. Iron in human atheroma and LDL oxidation by macrophages following erythrophagocytosis. Atherosclerosis. 1996 Jul;124(1):61–73. doi: 10.1016/0021-9150(96)05817-0. [DOI] [PubMed] [Google Scholar]
  51. Yuan X. M., Brunk U. T., Olsson A. G. Effects of iron- and hemoglobin-loaded human monocyte-derived macrophages on oxidation and uptake of LDL. Arterioscler Thromb Vasc Biol. 1995 Sep;15(9):1345–1351. doi: 10.1161/01.atv.15.9.1345. [DOI] [PubMed] [Google Scholar]
  52. Zwilling B. S., Kuhn D. E., Wikoff L., Brown D., Lafuse W. Role of iron in Nramp1-mediated inhibition of mycobacterial growth. Infect Immun. 1999 Mar;67(3):1386–1392. doi: 10.1128/iai.67.3.1386-1392.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. de Chastellier C., Fréhel C., Offredo C., Skamene E. Implication of phagosome-lysosome fusion in restriction of Mycobacterium avium growth in bone marrow macrophages from genetically resistant mice. Infect Immun. 1993 Sep;61(9):3775–3784. doi: 10.1128/iai.61.9.3775-3784.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

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