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Bryan Mackenzie - One of the best experts on this subject based on the ideXlab platform.
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Intestinal divalent metal-Ion Transporter-1 is critical for intestinal iron absorptIon but not that of copper (996.3)
The FASEB Journal, 2014Co-Authors: Ali Shawki, Sarah Anthony, Melinda A. Engevik, Eric Niespodzany, Roger T. Worrell, Bryan MackenzieAbstract:Divalent metal-Ion Transporter-1 (DMT1) is a widely expressed iron Transporter serving iron absorptIon and erythroid iron uptake. DMT1 can also transport certain other transitIon metal Ions; howeve...
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substrate profile and metal Ion selectivity of human divalent metal Ion Transporter 1
Journal of Biological Chemistry, 2012Co-Authors: Anthony C Illing, Ali Shawki, Christopher L Cunningham, Bryan MackenzieAbstract:Abstract 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 Imax/K0.5 (determined from evoked currents at −70 mV): Cd2+ > Fe2+ > Co2+, Mn2+ ≫ Zn2+, Ni2+, VO2+. DMT1 expressIon did not stimulate the transport of Cr2+, Cr3+, Cu+, Cu2+, Fe3+, Ga3+, Hg2+, or VO+. 55Fe2+ transport was competitively inhibited by Co2+ and Mn2+. Zn2+ only weakly inhibited 55Fe2+ transport. Our data reveal that DMT1 selects Fe2+ 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.
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h coupled divalent metal Ion Transporter 1 functIonal properties physiological roles and therapeutics
Current Topics in Membranes, 2012Co-Authors: Ali Shawki, Patrick B Knight, Bryan D Maliken, Eric J Niespodzany, Bryan MackenzieAbstract:Divalent metal-Ion Transporter-1 (DMT1) is a widely expressed, iron-preferring membrane transport protein. Animal models establish that DMT1 plays indispensable roles in intestinal nonheme-iron absorptIon and iron acquisitIon by erythroid precursor cells. Rare mutatIons in human DMT1 result in severe microcytic–hypochromic anemia. When we express DMT1 in RNA-injected Xenopus oocytes, we observe rheogenic Fe2+ transport that is driven by the proton electrochemical potential gradient. In that same preparatIon, DMT1 also transports cadmium and manganese but not copper. Whether manganese metabolism relies upon DMT1 remains unclear but DMT1 contributes to the effects of overexposure to cadmium and manganese in some tissues. There exist at least four DMT1 isoforms that arise from variant transcriptIon of the SLC11A2 gene. Whereas these isoforms display identical functIonal properties, N- and C-terminal variatIons contain cues that direct the cell-specific targeting of DMT1 isoforms to discrete subcellular compartments (plasma membrane, endosomes, and lysosomes). An iron-responsive element (IRE) in the mRNA 3′-untranslated regIon permits the regulatIon of some isoforms by iron status, and additIonal mechanisms by which DMT1 is regulated are emerging. Natural-resistance-associated macrophage protein-1 (NRAMP1)—the only other member of the mammalian SLC11 gene family—contributes to antimicrobial functIon by extruding from the phagolysosome divalent metal Ions (e.g. Mn2+) that may be essential cofactors for bacteria-derived enzymes or required for bacterial growth. The principal or only intestinal nonheme-iron Transporter, DMT1 is a validated therapeutic target in hereditary hemochromatosis (HHC) and other iron-overload disorders.
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Calcium-channel blockers do not affect iron transport mediated by divalent metal-Ion Transporter-1
Blood, 2010Co-Authors: Bryan Mackenzie, Ali Shawki, Andrew J. Ghio, Jacqueline D. Stonehuerner, Lin Zhao, Saied Ghadersohi, Laura M. Garrick, Michael D. GarrickAbstract:To the editor: Ludwiczek et al[1][1] reported that nifedipine and other dihydropyridine-class calcium-channel blockers can reverse iron overload by stimulating the activity of divalent metal-Ion Transporter-1 (DMT1)[2][2],[3][3] and consequently mobilizing tissue iron. Since their paper disagreed
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interactIon of calcium with the human divalent metal Ion Transporter 1
Biochemical and Biophysical Research Communications, 2010Co-Authors: Ali Shawki, Bryan MackenzieAbstract:Iron deficiency is the most prevalent micronutrient deficiency worldwide. Whereas dietary calcium is known to reduce the bioavailability of iron, the molecular basis of this interactIon is not understood. We tested the hypothesis that divalent metal-Ion Transporter-1 (DMT1)-the principal or only mechanism by which nonheme iron is taken up at the intestinal brush border-is shared also by calcium. We expressed human DMT1 in RNA-injected Xenopus oocytes and examined its activity using radiotracer assays and the voltage clamp. DMT1 did not mediate {sup 45}Ca{sup 2+} uptake. Instead, we found that Ca{sup 2+} blocked the Fe{sup 2+}-evoked currents and inhibited {sup 55}Fe{sup 2+} uptake in a noncompetitive manner (K{sub i} {approx} 20 mM). The mechanism of inhibitIon was independent of voltage and did not involve intracellular Ca{sup 2+} signaling. The alkaline-earth metal Ions Ba{sup 2+}, Sr{sup 2+}, and Mg{sup 2+} also inhibited DMT1-mediated iron-transport activity. We conclude that Ca{sup 2+} is a low-affinity noncompetitive inhibitor-but not a transported substrate-of DMT1, explaining in part the effect of high dietary calcium on iron bioavailability.
Matthias A Hediger - One of the best experts on this subject based on the ideXlab platform.
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functIonal properties of multiple isoforms of human divalent metal Ion Transporter 1 dmt1
Biochemical Journal, 2007Co-Authors: Bryan Mackenzie, Hitomi Takanaga, Nadia Hubert, Andreas Rolfs, Matthias A HedigerAbstract:DMT1 (divalent metal-Ion Transporter 1) is a widely expressed metal-Ion Transporter that is vital for intestinal iron absorptIon and iron utilizatIon by most cell types throughout the body, including erythroid precursors. MutatIons in DMT1 cause severe microcytic anaemia in animal models. Four DMT1 isoforms that differ in their N- and C-termini arise from mRNA transcripts that vary both at their 5'-ends (starting in exon 1A or exon 1B) and at their 3'-ends giving rise to mRNAs containing (+) or lacking (-) the 3'-IRE (iron-responsive element) and resulting in altered C-terminal coding sequences. To determine whether these variatIons result in functIonal differences between isoforms, we explored the functIonal properties of each isoform using the voltage clamp and radiotracer assays in cRNA-injected Xenopus oocytes. 1A/IRE+-DMT1 mediated Fe2+-evoked currents that were saturable (K(0.5)(Fe) approximately 1-2 microM), temperature-dependent (Q10 approximately 2), H+-dependent (K(0.5)(H) approximately 1 muM) and voltage-dependent. 1A/IRE+-DMT1 exhibited the provisIonal substrate profile (ranked on currents) Cd2+, Co2+, Fe2+, Mn2+>Ni2+, V3+>>Pb2+. Zn2+ also evoked large currents; however, the zinc-evoked current was accounted for by H+ and Cl- conductances and was not associated with significant Zn2+ transport. 1B/IRE+-DMT1 exhibited the same substrate profile, Fe2+ affinity and dependence on the H+ electrochemical gradient. Each isoform mediated 55Fe2+ uptake and Fe2+-evoked currents at low extracellular pH. Whereas iron transport activity varied markedly between the four isoforms, the activity for each correlated with the density of anti-DMT1 immunostaining in the plasma membrane, and the turnover rate of the Fe2+ transport cycle did not differ between isoforms. Therefore all four isoforms of human DMT1 functIon as metal-Ion Transporters of equivalent efficiency. Our results reveal that the N- and C-terminal sequence variatIons among the DMT1 isoforms do not alter DMT1 functIonal properties. We therefore propose that these variatIons serve as tissue-specific signals or cues to direct DMT1 to the appropriate subcellular compartments (e.g. in erythroid cells) or the plasma membrane (e.g. in intestine).
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divalent metal Ion Transporter dmt1 mediates both h coupled fe2 transport and uncoupled fluxes
Pflügers Archiv: European Journal of Physiology, 2006Co-Authors: Bryan Mackenzie, Michael F Romero, M L Ujwal, Min Hwang Chang, Matthias A HedigerAbstract: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 (pH(o)), H(+) binding preceded binding of Fe(2+) and its simultaneous translocatIon. However, DMT1 did not behave like a typical Ion-coupled Transporter at higher pH(o), and at pH(o) 7.4 we observed Fe(2+) transport that was not associated with H(+) influx. His(272) --> Ala substitutIon uncoupled the Fe(2+) and H(+) fluxes. At low pH(o), H272A mediated H(+) uniport that was inhibited by Fe(2+). Meanwhile H272A-mediated Fe(2+) transport was independent of pH(o). Our data indicate (i) that H(+) coupling in DMT1 serves to increase affinity for Fe(2+) and provide a thermodynamic driving force for Fe(2+) 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 Fe(2+) 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 Fe(2+) transport via DMT1 can account for the uptake of nontransferrin-bound plasma iron characteristic of iron overload disorders.
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SLC11 family of H + -coupled metal-Ion Transporters NRAMP1 and DMT1
Pflügers Archiv: European Journal of Physiology, 2003Co-Authors: Bryan Mackenzie, Matthias A HedigerAbstract:NRAMP1 (natural resistance-associated macrophage protein-1) and DMT1 (divalent metal-Ion Transporter-1) make up the SLC11 gene family of metal-Ion Transporters that are energized by the H+ electrochemical gradient. Long known to confer resistance to bacterial infectIon, NRAMP1 functIons at the phagolysosomal membrane of macrophages and neutrophils. NRAMP1 most likely contributes to macrophage antimicrobial functIon by extruding essential metal Ions (including Mn2+) from the phagolysosome via H+/metal-Ion cotransport. An alternative hypothesis in the literature proposes that NRAMP1 concentrate Fe2+ within the phagolysosome by means of H+/Fe2+ antiport, resulting in the generatIon of toxic free radicals. DMT1 is expressed widely and accepts as substrates a broad range of transitIon metal Ions, among which Fe2+ is transported with high affinity (K 0.5≈2 μM). DMT1 accounts both for the intestinal absorptIon of free Fe2+ and for transferrin-associated endosomal Fe2+ transport in erythroid precursors and many other cell types. DMT1 is up-regulated dramatically in the intestine by dietary iron restrictIon and, despite high serum iron levels, is not appropriately down-regulated in hereditary hemochromatosis.
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cloning and characterizatIon of a mammalian proton coupled metal Ion Transporter
Nature, 1997Co-Authors: Hiromi Gunshin, Bryan Mackenzie, Urs V Berger, Yoshimi Gunshin, Michael F Romero, Walter F Boron, Stephan Nussberger, John L Gollan, Matthias A HedigerAbstract: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 haemochromatosis1, disorders of metal-Ion deficiency, and certain neurodegenerative diseases2. 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) family3,4,5 and thus its properties provide insight into how these proteins confer resistance to pathogens.
Ali Shawki - One of the best experts on this subject based on the ideXlab platform.
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Intestinal divalent metal-Ion Transporter-1 is critical for intestinal iron absorptIon but not that of copper (996.3)
The FASEB Journal, 2014Co-Authors: Ali Shawki, Sarah Anthony, Melinda A. Engevik, Eric Niespodzany, Roger T. Worrell, Bryan MackenzieAbstract:Divalent metal-Ion Transporter-1 (DMT1) is a widely expressed iron Transporter serving iron absorptIon and erythroid iron uptake. DMT1 can also transport certain other transitIon metal Ions; howeve...
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substrate profile and metal Ion selectivity of human divalent metal Ion Transporter 1
Journal of Biological Chemistry, 2012Co-Authors: Anthony C Illing, Ali Shawki, Christopher L Cunningham, Bryan MackenzieAbstract:Abstract 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 Imax/K0.5 (determined from evoked currents at −70 mV): Cd2+ > Fe2+ > Co2+, Mn2+ ≫ Zn2+, Ni2+, VO2+. DMT1 expressIon did not stimulate the transport of Cr2+, Cr3+, Cu+, Cu2+, Fe3+, Ga3+, Hg2+, or VO+. 55Fe2+ transport was competitively inhibited by Co2+ and Mn2+. Zn2+ only weakly inhibited 55Fe2+ transport. Our data reveal that DMT1 selects Fe2+ 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.
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h coupled divalent metal Ion Transporter 1 functIonal properties physiological roles and therapeutics
Current Topics in Membranes, 2012Co-Authors: Ali Shawki, Patrick B Knight, Bryan D Maliken, Eric J Niespodzany, Bryan MackenzieAbstract:Divalent metal-Ion Transporter-1 (DMT1) is a widely expressed, iron-preferring membrane transport protein. Animal models establish that DMT1 plays indispensable roles in intestinal nonheme-iron absorptIon and iron acquisitIon by erythroid precursor cells. Rare mutatIons in human DMT1 result in severe microcytic–hypochromic anemia. When we express DMT1 in RNA-injected Xenopus oocytes, we observe rheogenic Fe2+ transport that is driven by the proton electrochemical potential gradient. In that same preparatIon, DMT1 also transports cadmium and manganese but not copper. Whether manganese metabolism relies upon DMT1 remains unclear but DMT1 contributes to the effects of overexposure to cadmium and manganese in some tissues. There exist at least four DMT1 isoforms that arise from variant transcriptIon of the SLC11A2 gene. Whereas these isoforms display identical functIonal properties, N- and C-terminal variatIons contain cues that direct the cell-specific targeting of DMT1 isoforms to discrete subcellular compartments (plasma membrane, endosomes, and lysosomes). An iron-responsive element (IRE) in the mRNA 3′-untranslated regIon permits the regulatIon of some isoforms by iron status, and additIonal mechanisms by which DMT1 is regulated are emerging. Natural-resistance-associated macrophage protein-1 (NRAMP1)—the only other member of the mammalian SLC11 gene family—contributes to antimicrobial functIon by extruding from the phagolysosome divalent metal Ions (e.g. Mn2+) that may be essential cofactors for bacteria-derived enzymes or required for bacterial growth. The principal or only intestinal nonheme-iron Transporter, DMT1 is a validated therapeutic target in hereditary hemochromatosis (HHC) and other iron-overload disorders.
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Calcium-channel blockers do not affect iron transport mediated by divalent metal-Ion Transporter-1
Blood, 2010Co-Authors: Bryan Mackenzie, Ali Shawki, Andrew J. Ghio, Jacqueline D. Stonehuerner, Lin Zhao, Saied Ghadersohi, Laura M. Garrick, Michael D. GarrickAbstract:To the editor: Ludwiczek et al[1][1] reported that nifedipine and other dihydropyridine-class calcium-channel blockers can reverse iron overload by stimulating the activity of divalent metal-Ion Transporter-1 (DMT1)[2][2],[3][3] and consequently mobilizing tissue iron. Since their paper disagreed
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interactIon of calcium with the human divalent metal Ion Transporter 1
Biochemical and Biophysical Research Communications, 2010Co-Authors: Ali Shawki, Bryan MackenzieAbstract:Iron deficiency is the most prevalent micronutrient deficiency worldwide. Whereas dietary calcium is known to reduce the bioavailability of iron, the molecular basis of this interactIon is not understood. We tested the hypothesis that divalent metal-Ion Transporter-1 (DMT1)-the principal or only mechanism by which nonheme iron is taken up at the intestinal brush border-is shared also by calcium. We expressed human DMT1 in RNA-injected Xenopus oocytes and examined its activity using radiotracer assays and the voltage clamp. DMT1 did not mediate {sup 45}Ca{sup 2+} uptake. Instead, we found that Ca{sup 2+} blocked the Fe{sup 2+}-evoked currents and inhibited {sup 55}Fe{sup 2+} uptake in a noncompetitive manner (K{sub i} {approx} 20 mM). The mechanism of inhibitIon was independent of voltage and did not involve intracellular Ca{sup 2+} signaling. The alkaline-earth metal Ions Ba{sup 2+}, Sr{sup 2+}, and Mg{sup 2+} also inhibited DMT1-mediated iron-transport activity. We conclude that Ca{sup 2+} is a low-affinity noncompetitive inhibitor-but not a transported substrate-of DMT1, explaining in part the effect of high dietary calcium on iron bioavailability.
Mitchell D Knutson - One of the best experts on this subject based on the ideXlab platform.
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the plasma membrane metal Ion Transporter zip14 contributes to nontransferrin bound iron uptake by human β cells
American Journal of Physiology-cell Physiology, 2017Co-Authors: Richard Coffey, Mitchell D KnutsonAbstract:The relatIonship between iron and β-cell dysfunctIon has long been recognized as individuals with iron overload display an increased incidence of diabetes. This link is usually attributed to the accumulatIon of excess iron in β-cells leading to cellular damage and impaired functIon. Yet, the molecular mechanism(s) by which human β-cells take up iron has not been determined. In the present study, we assessed the contributIon of the metal-Ion Transporters ZRT/IRT-like protein 14 and 8 (ZIP14 and ZIP8) and divalent metal-Ion Transporter-1 (DMT1) to iron uptake by human β-cells. Iron was provided to the cells as nontransferrin-bound iron (NTBI), which appears in the plasma during iron overload and is a major contributor to tissue iron loading. We found that overexpressIon of ZIP14 and ZIP8, but not DMT1, resulted in increased NTBI uptake by βlox5 cells, a human β-cell line. Conversely, siRNA-mediated knockdown of ZIP14, but not ZIP8, resulted in 50% lower NTBI uptake in βlox5 cells. In primary human islets, knockdown of ZIP14 also reduced NTBI uptake by 50%. Immunofluorescence analysis of islets from human pancreatic sectIons localized ZIP14 and DMT1 nearly exclusively to β-cells. Studies in primary human islets suggest that ZIP14 protein levels do not vary with iron status or treatment with IL-1β. Collectively, these observatIons identify ZIP14 as a major contributor to NTBI uptake by β-cells and suggest differential regulatIon of ZIP14 in primary human islets compared with other cell types such as hepatocytes.
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hepatocyte divalent metal Ion Transporter 1 is dispensable for hepatic iron accumulatIon and non transferrin bound iron uptake in mice
Hepatology, 2013Co-Authors: Chiayu Wang, Mitchell D KnutsonAbstract:Divalent metal-Ion Transporter-1 (DMT1) is required for iron uptake by the intestine and developing erythroid cells. DMT1 is also present in the liver, where it has been implicated in the uptake of transferrin-bound iron (TBI) and non-transferrin-bound iron (NTBI), which appears in the plasma during iron overload. To test the hypothesis that DMT1 is required for hepatic iron uptake, we examined mice with the Dmt1 gene selectively inactivated in hepatocytes (Dmt1liv/liv). We found that Dmt1liv/liv mice and controls (Dmt1flox/flox) did not differ in terms of hepatic iron concentratIons or other parameters of iron status. To determine if hepatocyte DMT1 is required for hepatic iron accumulatIon, we crossed Dmt1liv/liv mice with Hfe−/− and hypotransferrinemic (Trfhpx/hpx) mice that develop hepatic iron overload. Double-mutant Hfe−/−;Dmt1liv/liv and Trfhpx/hpx;Dmt1liv/liv mice were found to accumulate similar amounts of hepatic iron as did their respective controls. To directly assess the role of DMT1 in NTBI and TBI uptake, we injected 59Fe-labeled ferric citrate (for NTBI) or 59Fe-transferrin into the plasma of Dmt1liv/liv and Dmt1flox/flox mice and measured the uptake of 59Fe by the liver. Dmt1liv/liv mice displayed no impairment of hepatic NTBI uptake, but TBI uptake was 40% lower. Hepatic levels of transferrin receptors 1 and 2 and ZIP14 (ZRT/IRT-like protein 14), which may also participate in iron uptake, were unaffected in Dmt1liv/liv mice. AdditIonally, liver iron levels were unaffected in Dmt1liv/liv mice fed an iron-deficient diet. ConclusIon Hepatocyte DMT1 is dispensable for hepatic iron accumulatIon and NTBI uptake. Although hepatocyte DMT1 is partially required for hepatic TBI uptake, hepatic iron levels were unaffected in Dmt1liv/liv mice, suggesting that this pathway is a minor contributor to the iron economy of the liver.
Jack H. Kaplan - One of the best experts on this subject based on the ideXlab platform.
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dynamic internalizatIon and recycling of a metal Ion Transporter cu homeostasis and ctr1 the human cu uptake system
Journal of Cell Science, 2016Co-Authors: Rebecca J. Clifford, Edward B. Maryon, Jack H. KaplanAbstract:Cu Ion (Cu) entry into human cells is mediated by CTR1 (also known as SLC31A1), the high-affinity Cu Transporter. When extracellular Cu is raised, the cell is protected against excess accumulatIon by rapid internalizatIon of the Transporter. When Cu is lowered, the Transporter returns to the membrane. We show in HEK293 cells overexpressing CTR1 that expressIon of either the C-terminal domain of AP180 (also known as SNAP91), a clathrin-coat assembly protein that sequesters clathrin, or a dominant-negative mutant of dynamin, decreases Cu-induced endocytosis of CTR1, as does a dynamin inhibitor and clathrin knockdown using siRNA. Utilizing imaging, siRNA techniques and a new high-throughput assay for endocytosis employing CLIP-tag methodology, we show that internalized CTR1 accumulates in early sorting endosomes and recycling compartments (containing Rab5 and EEA1), but not in late endosomes or lysosomal pathways. Using live cell fluorescence, we find that upon extracellular Cu removal CTR1 recycles to the cell surface through the slower-recycling Rab11-mediated pathway. These processes enable cells to dynamically alter Transporter levels at the plasma membrane and acutely modulate entry as a safeguard against excess cellular Cu.
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Dynamic internalizatIon and recycling of a metal Ion Transporter: Cu homeostasis and CTR1, the human Cu+ uptake system
Journal of Cell Science, 2016Co-Authors: Rebecca J. Clifford, Edward B. Maryon, Jack H. KaplanAbstract:Cu Ion (Cu) entry into human cells is mediated by CTR1 (also known as SLC31A1), the high-affinity Cu Transporter. When extracellular Cu is raised, the cell is protected against excess accumulatIon by rapid internalizatIon of the Transporter. When Cu is lowered, the Transporter returns to the membrane. We show in HEK293 cells overexpressing CTR1 that expressIon of either the C-terminal domain of AP180 (also known as SNAP91), a clathrin-coat assembly protein that sequesters clathrin, or a dominant-negative mutant of dynamin, decreases Cu-induced endocytosis of CTR1, as does a dynamin inhibitor and clathrin knockdown using siRNA. Utilizing imaging, siRNA techniques and a new high-throughput assay for endocytosis employing CLIP-tag methodology, we show that internalized CTR1 accumulates in early sorting endosomes and recycling compartments (containing Rab5 and EEA1), but not in late endosomes or lysosomal pathways. Using live cell fluorescence, we find that upon extracellular Cu removal CTR1 recycles to the cell surface through the slower-recycling Rab11-mediated pathway. These processes enable cells to dynamically alter Transporter levels at the plasma membrane and acutely modulate entry as a safeguard against excess cellular Cu.
