Abstract
The H+ -coupled divalent metal-ion transporter DMT1 serves as both the primary entry point for iron into the body (intestinal brush-border uptake) and the route by which transferrin-associated iron is mobilized from endosomes to cytosol in erythroid precursors and other cells. Elucidating the molecular mechanisms of DMT1 will therefore increase our understanding of iron metabolism and the etiology of iron overload disorders. We expressed wild type and mutant DMT1 in Xenopus oocytes and monitored metal-ion uptake, currents and intracellular pH. DMT1 was activated in the presence of an inwardly directed H+ electrochemical gradient. At low extracellular pH (pHo), H+ binding preceded binding of Fe2+ and its simultaneous translocation. However, DMT1 did not behave like a typical ion-coupled transporter at higher pHo, and at pHo 7.4 we observed Fe2+ transport that was not associated with H+ influx. His272 → Ala substitution uncoupled the Fe2+ and H+ fluxes. At low pHo, H272A mediated H+ uniport that was inhibited by Fe2+. Meanwhile H272A-mediated Fe2+ transport was independent of pHo. Our data indicate (i) that H+ coupling in DMT1 serves to increase affinity for Fe2+ and provide a thermodynamic driving force for Fe2+ transport and (ii) that His-272 is critical in transducing the effects of H+ coupling. Notably, our data also indicate that DMT1 can mediate facilitative Fe2+ transport in the absence of a H+ gradient. Since plasma membrane expression of DMT1 is upregulated in liver of hemochromatosis patients, this H+ -uncoupled facilitative Fe2+ transport via DMT1 can account for the uptake of nontransferrin-bound plasma iron characteristic of iron overload disorders.
Similar content being viewed by others
References
Aslamkhan AG, Aslamkhan A, Ahearn GA (2002) Preparation of metal ion buffers for biological experimentation: a methods approach with emphasis on iron and zinc. J Exp Biol 292:507–522
Bannon DI, Abounader R, Lees PS, Bressler JP (2003) Effect of DMT1 knockdown on iron, cadmium, and lead uptake in Caco-2 cells. Am J Physiol Cell Physiol 284:C44–C50
Becker HM, Bröer S, Deitmer JW (2004) Facilitated lactate transport by MCT1 when coexpressed with the sodium bicarbonate cotransporter (NBC) in Xenopus oocytes. Biophys J 86:235–247
Canonne-Hergaux F, Gros P (2002) Expression of the iron transporter DMT1 in kidney from normal and anemic mk mice. Kidney Int 62:147–156
Canonne-Hergaux F, Gruenheid S, Ponka P, Gros P (1999) Cellular and subcellular localization of the Nramp2 iron transporter in the intestinal brush border and regulation by dietary iron. Blood 93:4406–4417
Canonne-Hergaux F, Zhang A-S, Ponka P, Gros P (2001) Characterization of the iron transporter DMT1 (NRAMP2/DCT1) in red blood cells of normal and anemic mk/mk mice. Blood 98:3823–3830
Chen X-Z, Peng J-B, Cohen A, Nelson H, Nelson N, Hediger MA (1999) Yeast SMF1 mediates H+ -coupled iron uptake with concomitant uncoupled cation currents. J Biol Chem 274:35089–35094
de Valk B, Addicks MA, Gosriwatana I, Lu S, Hider RC, Marx JJ (2000) Non-transferrin-bound iron is present in serum of hereditary haemochromatosis heterozygotes. Eur J Clin Invest 30:248–251
Diez-Sampedro A, Lostao MP, Wright EM, Hirayama BA (2000) Glycoside binding and translocation in Na+ -dependent glucose cotransporters: comparison of SGLT1 and SGLT3. J Membr Biol 176:111–117
Dinour D, Chang M-H, Satoh J, Smith BL, Angle N, Knecht A, Serban I, Holtzman EJ, Romero MF (2004) A novel missense mutation in the sodium bicarbonate cotransporter (NBCe1/SLC4A4) causes proximal tubular acidosis and glaucoma through ion transport defects. J Biol Chem 279:52238–52246
Fischer BE, Haring UK, Tribolet R, Sigel H (1979) Metal ion/buffer interactions: stability of binary and ternary complexes containing 2-amino-2(hydroxymethyl)-1,3-propanediol (Tris) and adenosine 5′-triphosphate (ATP). Eur J Biochem 94:523–530
Fleming MD, Romano MA, Su MA, Garrick LM, Garrick MD, Andrews NC (1998) Nramp2 is mutated in the anemic Belgrade (b) rat: evidence of a role for Nramp2 in endosomal iron transport. Proc Natl Acad Sci USA 95:1148–1153
Fleming MD, Trenor CC, Su MA, Foernzler D, Beier DR, Dietrich WF, Andrews NC (1997) Microcytic anaemia mice have a mutation in Nramp2, a candidate iron transporter gene. Nat Genet 16:383–386
Forbes JR, Gros P (2003) Iron, manganese, and cobalt transport by Nramp1 (Slc11a1) and Nramp2 (Slc11a2) expressed at the plasma membrane. Blood 102:1884–1892
Garrick LM, Dolan KG, Romano MA, Garrick MD (1999) Non-transferrin-bound iron uptake in Belgrade and normal rat erythroid cells. J Cell Physiol 178:349–358
Garrick MD, Dolan KG, Horbinski C, Ghio AJ, Higgins D, Porubcin M, Moore EG, Hainsworth LN, Umbreit JN, Conrad ME, Feng L, Lis A, Roth JA, Singleton S, Garrick LM (2003) DMT1: a mammalian transporter for multiple metals. Biometals 16:41–54
Grootveld M, Bell JD, Halliwell B, Aruoma OI, Bomford A, Sadler PJ (1989) Non-transferrin-bound iron in plasma or serum from patients with idiopathic hemochromatosis. Characterization by high performance liquid chromatography and nuclear magnetic resonance spectroscopy. J Biol Chem 264:4417–4422
Gruenheid S, Canonne-Hergaux F, Gauthier S, Hackam DJ, Grinstein S, Gros P (1999) The iron transport protein NRAMP2 is an integral membrane glycoprotein that colocalizes with transferrin in recycling endosomes. J Exp Med 189:831–841
Gunshin H, Mackenzie B, Berger UV, Gunshin Y, Romero MF, Boron WF, Nussberger S, Gollan JL, Hediger MA (1997) Cloning and characterization of a proton-coupled mammalian metal-ion transporter. Nature 388:482–488
Harrison SA, Bacon BR (2003) Hereditary hemochromatosis: update for 2003. J Hepatol 38:S14–S23
Hazama A, Loo DDF, Wright EM (1997) Presteady-state currents of the rabbit Na+/glucose cotransporter (SGLT1). J Membr Biol 155:175–186
Hershko C, Graham G, Bates GW, Rachmilewitz EA (1978) Non-specific serum iron in thalassaemia: an abnormal serum iron fraction of potential toxicity. Br J Haematol 40:255–263
Hubert N, Hentze MW (2002) Previously uncharacterized isoforms of divalent metal transporter (DMT)-1: implications for regulation and cellular function. Proc Natl Acad Sci USA 99:12345–12350
Inman RS, Coughlan MM, Wessling-Resnick M (1994) Extracellular ferrireductase activity of K562 cells is coupled to transferrin-independent iron transport. Biochemistry 33:11850–11857
Jauch P, Läuger P (1986) Electrogenic properties of the sodium-alanine cotransporter in pancreatic acinar cells: II. Comparison with transport models. J Membr Biol 94:117–127
Jordan I, Kaplan J (1994) The mammalian transferrin-independent iron transport system may involve a surface ferrireductase activity. Biochem J 302:875–879
Ke Y, Chen YY, Chang YZ, Duan XL, Ho KP, Jiang DH, Wang K, Qian ZM (2003) Post-transcriptional expression of DMT1 in the heart of rat. J Cell Physiol 196:124–130
Klamo EM, Drew ME, Landfear SM, Kavanaugh MP (1996) Kinetics and stoichiometry of a proton/myo-inositol cotransporter. J Biol Chem 271:14937–14943
Lam-Yuk-Tseung S, Govoni G, Gros P (2003) Iron transport by NRAMP2/DMT1: pH regulation of transport by two histidines in transmembrane domain 6. Blood 101:3699–3707
Loo DDF, Hazama A, Supplisson S, Turk E, Wright EM (1993) Relaxation kinetics of the Na+/glucose cotransporter. Proc Natl Acad Sci USA 90:5767–5771
Lostao MP, Hirayama BA, Loo DDF, Wright EM (1994) Phenylglucosides and the Na+/glucose cotransporter (SGLT1): analysis of interactions. J Membr Biol 142:161–170
Mackenzie B (1999) Selected techniques in membrane transport. In: Van Winkle LJ (ed) Biomembrane transport. Academic Press, San Diego, pp 327–342
Mackenzie B, Hediger MA (2004) SLC11 family of H+ -coupled metal-ion transporters NRAMP1 and DMT1. Pflügers Arch Eur J Physiol 447:571–579
Mackenzie B, Loo DDF, Fei YJ, Liu W, Ganapathy V, Leibach FH, Wright EM (1996) Mechanisms of the human intestinal H+ -coupled oligopeptide transporter hPEPT1. J Biol Chem 271:5430–5437
Mackenzie B, Loo DDF, Panayotova-Heiermann M, Wright EM (1996) Biophysical characteristics of the pig kidney Na+/glucose cotransporter SGLT2 reveal a common mechanism for SGLT1 and SGLT2. J Biol Chem 271:32678–32683
Mackenzie B, Schäfer MKH, Erickson JD, Hediger MA, Weihe E, Varoqui H (2003) Functional properties and cellular distribution of the System A glutamine transporter SNAT1 support specialized roles in central neurons. J Biol Chem 278:23720–23730
McEwan GTA, Daniel H, Fett C, Burgess MN, Lucas ML (1988) The effect of Escherichia coli STa enterotoxin and other secretagogues on mucosal surface pH of rat small intestine in vivo. Proc R Soc Lond B 234:219–237
McEwan GTA, Lucas ML, Mathan VI (1990) A combined TDDA-PVC pH and reference electrode for use in the upper small intestine. J Med Eng Tech 14:16–20
Moridani MY, O’Brien PJ (2001) Iron complexes of deferiprone and dietary plant catechols as cytoprotective superoxide radical scavengers (1). Biochem Pharmacol 62:1579–1585
Núñez M-T, Gaete V, Watkins JA, Glass J (1990) Mobilization of iron from endocytic vesicles. The effects of acidification and reduction. J Biol Chem 265:6688–6692
Núñez M-T, Gaete V, Watkins JA, Glass J (1990) Mobilization of iron from endocytic vesicles. The effects of acidification and reduction. J Biol Chem 265:6688–6692
Olivieri NF (2002) Transfusional iron overload. In: Templeton DM (ed) Molecular and cellular iron transport. Marcel-Dekker, New York, pp 725–747
Parent L, Supplisson S, Loo DDF, Wright EM (1992) Electrogenic properties of the cloned Na+/glucose cotransporter: I. Voltage-clamp studies. J Membr Biol 125:49–62
Parent L, Supplisson S, Loo DDF, Wright EM (1992) Electrogenic properties of the cloned Na+/glucose cotransporter: II. A transport model under nonrapid equilibrium conditions. J Membr Biol 125:63–79
Picard V, Govoni G, Jabado N, Gros P (2000) Nramp 2 (DCT1/DMT1) expressed at the plasma membrane transports iron and other divalent cations into a calcein-accessible cytoplasmic pool. J Biol Chem 275:35738–35745
Rawlings JM, Lucas ML, Russell RI (1987) Measurement of jejunal surface pH in situ by plastic pH electrode in patients with coeliac disease. Scand J Gastroenterol 22:377–384
Said HM, Tipton W, Nylander W, Urban E (1987) Effect of small bowel resection on the intestinal surface acid microclimate in the rat. Digestion 38:221–225
Shimada T, Hoshi T (1988) Na+ -dependent elevation of the acidic cell surface pH (microclimate pH) of rat jejunal villus cells induced by cyclic nucleotides and phorbol ester: possible mediators of the regulation of the Na+/H+ antiporter. Biochim Biophys Acta 937:328–334
Stein WD (1986) Transport and diffusion across cell membranes. Academic Press, Orlando
Su MA, Trenor CC, Fleming JC, Fleming MD, Andrews NC (1998) The G185R mutation disrupts function of the iron transporter Nramp2. Blood 92:2157–2163
Tandy S, Williams M, Leggett A, Lopez-Jimenez M, Dedes M, Ramesh B, Srai SK, Sharp P (2000) Nramp2 expression is associated with pH-dependent iron uptake across the apical membrane of human intestinal Caco-2 cells. J Biol Chem 275:1023–1029
Touret N, Furuya W, Forbes J, Gros P, Grinstein S (2003) Dynamic traffic through the recycling compartment couples the metal transporter Nramp2 (DMT1) with the transferrin receptor. J Biol Chem 278:25548–25557
Trinder D, Oates PS, Thomas C, Sadleir J, Morgan EH (2000) Localisation of divalent metal transporter 1 (DMT1) to the microvillus membrane of rat duodenal enterocytes in iron deficiency, but to hepatocytes in iron overload. Gut 46:270–276
Trinder D, Morgan E (1998) Mechanisms of ferric citrate uptake by human hepatoma cells. Am J Physiol Gastrointest Liver Physiol 275:G279–G286
Wadiche JI, Arriza JL, Amara SG, Kavanaugh MP (1995) Kinetics of a human glutamate transporter. Neuron 14:1019–1027
Wareing M, Ferguson CJ, Green R, Riccardi D, Smith CP (2000) In vivo characterization of renal iron transport in the anaesthetized rat. J Physiol 524:581–586
Worthington MT, Browne L, Battle EH, Luo RQ (2000) Functional properties of transfected human DMT1 iron transporter. Am J Physiol Gastrointest Liver Physiol 279:G1265–G1273
Wright TL, Brissot P, Ma W-L, Weisiger RA (1986) Characterization of non-transferrin-bound iron clearance by rat liver. J Biol Chem 261:10909–10914
Xu H, Jin J, DeFelice LJ, Andrews NC, Clapham DE (2004) A spontaneous, recurrent mutation in divalent metal transporter-1 exposes a calcium entry pathway. PLoS Biol 2:E50
Yu Q, Kandegedara A, Xu Y, Rorabacher DB (1997) Avoiding interferences from Good’s buffers: A contiguous series of noncomplexing tertiary amine buffers covering the entire range of pH 3-11. Anal Biochem 253:50–56
Zampighi GA, Kreman M, Boorer KJ, Loo DDF, Bezanilla F, Chandy G, Hall JE, Wright EM (1995) A method for determining the unitary functional capacity of cloned channels and transporters expressed in Xenopus laevis oocytes. J Membr Biol 148:65–78
Acknowledgments
We are grateful to Hitomi Takanaga and Jonathan Sabbagh for their help in the laboratory, and to William A. Stein for advice regarding mathematical fitting. This study was supported by NIH grants R01-DK057782 (to M.A.H.) and R01-DK056218 (to M.F.R.), and a pilot/feasibility award (to B.M.) from the Harvard Digestive Diseases Center, funded by NIH center grant P30-DK034854.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Mackenzie, B., Ujwal, M.L., Chang, MH. et al. Divalent metal-ion transporter DMT1 mediates both H+ -coupled Fe2+ transport and uncoupled fluxes. Pflugers Arch - Eur J Physiol 451, 544–558 (2006). https://doi.org/10.1007/s00424-005-1494-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00424-005-1494-3