二价金属离子转运蛋白1——一个新发现的重要铁转运蛋白

二价金属离子转运蛋白1(divalent metal transporter 1,DMT1)的发现是近年铁代谢研究领域最重大的一项突破.DMT1是哺乳类跨膜铁转运蛋白.这种蛋白质广泛分布于人体各组织.DMT1 mRNA有两种形式,一种含有IRE(iron response element),而另一种则不含此结构.DMT1的功能主要是介导小肠上皮细胞的铁吸收以及参与铁从内吞小体移位到胞浆的过程.DMT1介导的铁转运是一个主动的和H⁺依赖的过程.DMT1也参与其他二价金属如Zn²⁺、Mn²⁺、Co²⁺、Cd²⁺、Cn²⁺、Ni²⁺和Pb²⁺的转运.小肠DMT1的表达受饮食或组织铁控制.第四跨膜区是DMT1的重要功能区.此区基因发生点突变（G185R）是导致不可逆性缺铁性贫血的原因.在帕金森氏病人的黑质发现DMT1表达异常增加,因而DMT1可能也与某些神经退行性疾病的形成有关.     

Dcytb (Cybrd1) functions as both a ferric and a cupric reductase in vitro

MDCK cells expressing an inducible duodenal cytochrome b-green fluorescent protein (Dcytb-EGFP) fusion construct were used to investigate the function of Dcytb. The Dcytb-EGFP protein was targeted correctly to the plasma membrane, and cells displayed increased ferric and cupric reductase activities, which were greatly reduced in the presence of doxycycline. The data suggests that Dcytb plays a physiological role in both iron and copper uptake, through divalent metal transporter 1 (DMT1) and copper transporter 1, respectively. In support of this hypothesis, we show that 59Fe uptake was significantly enhanced in Dcytb-EGFP expressing MDCK cells which endogenously express DMT1.     

Mitochondria represent another locale for the divalent metal transporter 1 (DMT1)

The divalent metal transporter (DMT1) is well known for its roles in duodenal iron absorption across the apical enterocyte membrane, in iron efflux from the endosome during transferrin-dependent cellular iron acquisition, as well as in uptake of non-transferrin bound iron in many cells. Recently, using multiple approaches, we have obtained evidence that the mitochondrial outer membrane is another subcellular locale of DMT1 expression. While iron is of vital importance for mitochondrial energy metabolism, its delivery is likely to be tightly controlled due to iron's damaging redox properties. Here we provide additional support for a role of DMT1 in mitochondrial iron acquisition by immunofluorescence colocalization with mitochondrial markers in cells and isolated mitochondria, as well as flow cytometric quantification of DMT1-positive mitochondria from an inducible expression system. Physiological consequences of mitochondrial DMT1 expression are discussed also in consideration of other DMT1 substrates, such as manganese, relevant to mitochondrial antioxidant defense.     

Mitochondria represent another locale for the divalent metal transporter 1 (DMT1)

The divalent metal transporter (DMT1) is well known for its roles in duodenal iron absorption across the apical enterocyte membrane, in iron efflux from the endosome during transferrin-dependent cellular iron acquisition, as well as in uptake of non-transferrin bound iron in many cells. Recently, using multiple approaches, we have obtained evidence that the mitochondrial outer membrane is another subcellular locale of DMT1 expression. While iron is of vital importance for mitochondrial energy metabolism, its delivery is likely to be tightly controlled due to iron''s damaging redox properties. Here we provide additional support for a role of DMT1 in mitochondrial iron acquisition by immunofluorescence colocalization with mitochondrial markers in cells and isolated mitochondria, as well as flow cytometric quantification of DMT1-positive mitochondria from an inducible expression system. Physiological consequences of mitochondrial DMT1 expression are discussed also in consideration of other DMT1 substrates, such as manganese, relevant to mitochondrial antioxidant defense.     

Enhanced autophagy from chronic toxicity of iron and mutant A53T α-synuclein: implications for neuronal cell death in Parkinson disease

Parkinson disease (PD), a prevalent neurodegenerative motor disorder, is characterized by the rather selective loss of dopaminergic neurons and the presence of α-synuclein-enriched Lewy body inclusions in the substantia nigra of the midbrain. Although the etiology of PD remains incompletely understood, emerging evidence suggests that dysregulated iron homeostasis may be involved. Notably, nigral dopaminergic neurons are enriched in iron, the uptake of which is facilitated by the divalent metal ion transporter DMT1. To clarify the role of iron in PD, we generated SH-SY5Y cells stably expressing DMT1 either singly or in combination with wild type or mutant α-synuclein. We found that DMT1 overexpression dramatically enhances Fe(2+) uptake, which concomitantly promotes cell death. This Fe(2+)-mediated toxicity is aggravated by the presence of mutant α-synuclein expression, resulting in increased oxidative stress and DNA damage. Curiously, Fe(2+)-mediated cell death does not appear to involve apoptosis. Instead, the phenomenon seems to occur as a result of excessive autophagic activity. Accordingly, pharmacological inhibition of autophagy reverses cell death mediated by Fe(2+) overloading. Taken together, our results suggest a role for iron in PD pathogenesis and provide a mechanism underlying Fe(2+)-mediated cell death.     

The effects of pH and the iron redox state on iron uptake in the intestine of a marine teleost fish, gulf toadfish (Opsanus beta)

In the marine teleost intestine the secretion of bicarbonate increases pH of the lumen (pH 8.4 -9.0) and importantly reduces Ca2+ and Mg2+ concentrations by the formation of insoluble divalent ion carbonates. The alkaline intestinal environment could potentially also cause essential metal carbonate formation reducing bioavailability. Iron accumulation was assessed in the Gulf toadfish (Opsanus beta) gut by mounting intestine segments in modified Ussing chambers fitted to a pH-stat titration system. This system titrates to maintain lumen pH constant and in the process prevents bicarbonate accumulation. The luminal saline pH was clamped to pH 5.5 or 7.0 to investigate the effect of proton concentrations on iron uptake. In addition, redox state was altered (gassing with N2, addition of dithiothreitol (DTT) and ascorbate) to evaluate Fe3+ versus Fe2+ uptake, enabling us to compare a marine teleost intestine model for iron uptake to the mammalian system for non-haem bound iron uptake that occurs via a ferrous/proton (Fe2+/H+) symporter called Divalent Metal Transporter 1 (DMT1). None of the redox altering strategies affected iron (Fe3+ or Fe2+) binding to mucus, but the addition of ascorbate resulted in a 4.6-fold increase in epithelium iron accumulation. This indicates that mucus iron binding is irrespective of valency and suggests that ferrous iron is preferentially transported across the apical surface. Altering luminal saline pH from 7.0 to 5.5 did not affect ferric or ferrous iron uptake, suggesting that if iron is entering via DMT1 in marine fish intestine this transporter works efficiently under circumneutral conditions.     

Uptake of lead and iron by divalent metal transporter 1 in yeast and mammalian cells

Although the divalent metal transporter (DMT1) was suggested to transport a wide range of metals in Xenopus oocytes, recent studies in other models have provided contrasting results. Here, we provide direct evidence demonstrating that DMT1 expressed in yeast mutants defective for high affinity iron transport facilitates the transport of iron with an 'apparent K(m)' of approximately 1.2 microM, and transport of lead with an 'apparent K(m)' of approximately 1.8 microM. DMT1-dependent lead transport was H(+)-dependent and was inhibited by iron. Human embryonic kidney fibroblasts (HEK293 cells) overexpressing DMT1 also showed a higher uptake of lead than HEK293 cells without overexpressing DMT1. These results show that DMT1 transports lead and iron with similar affinity in a yeast model suggesting that DMT1 is a transporter for lead.     

Correlation between the expression of divalent metal transporter 1 and the content of hypoxia-inducible factor-1 in hypoxic HepG2 cells

Transferrin and transferrin receptor are two key proteins of iron metabolism that have been identified to be hypoxia-inducible genes. Divalent metal transporter 1 (DMT1) is also a key transporter of iron under physiological conditions. In addition, in the 5' regulatory region of human DMT1 (between -412 and -570), there are two motifs (CCAAAGTGCTGGG) that are similar to hypoxia-inducible factor-1 (HIF-1) binding sites. It was therefore speculated that DMT1 might also be a hypoxia-inducible gene. We investigated the effects of hypoxia and hypoxia/re-oxygenation on the expression of DMT1 and the content of HIF-1alpha in HepG2 cells. As we expected, a very similar tendency in the responses of the expression of HIF-1alpha, DMT1+IRE (iron response element) and DMT1-IRE proteins to chemical (CoCl(2)) or physical hypoxia was observed. A highly significant correlation was found between the expression of DMT1 proteins and the contents of HIF-1 in hypoxic cells. After the cells were exposed to hypoxia and subsequent normoxia, no HIF-1alpha could be detected and a significant decrease in DMT1+IRE expression (P<0.05), but not in DMT1-IRE protein (versus the hypoxia group), was observed. The findings implied that the HIF-1 pathway might have a role in the regulation of DMT1+IRE expression during hypoxia.     

Regulation of divalent metal transporter expression in human intestinal epithelial cells following exposure to non-haem iron

A number of regulatory factors including dietary iron levels can dramatically alter the expression of the intestinal iron transporter DMT1. Here we show that Caco-2 cells exposed to iron for 4h exhibited a significant decrease in plasma membrane DMT1 protein, though total cellular DMT1 levels were unaltered. Following biotinylation of cell surface proteins, there was a significant increase in intracellular biotin-labelled DMT1 in iron-exposed cells. Furthermore, iron-treatment increased levels of DMT1 co-localised with LAMP1, suggesting that the initial response of intestinal epithelial cells to iron involves internalisation and targeting of DMT1 transporter protein towards a late endosomal/lysosomal compartment.     

Decreased DMT1 and increased ferroportin 1 expression is the mechanisms of reduced iron retention in macrophages by erythropoietin in rats

Recycled iron from reticuloendothelial macrophages to erythroid precursors is important to maintain the iron homeostasis. However, the molecular mechanisms underlying iron homeostasis in macrophages are poorly understood. In this study, male Sprague-Dawley rats were treated with recombinant human erythropoietin (rHuEpo, 500 IU/day, s.c.) for 3 days. At the fifth day, peritoneal exudate macrophages were harvested, and then (55)Fe uptake and release were measured by liquid scintillation counting method. The expression of divalent metal transporter 1 (DMT1) and ferroportin 1 (FPN1) in peritoneal exudate macrophages was detected by RT-PCR and Western blot. In order to exclude the direct effect of rHuEpo on macrophages, the parallel experiments were performed with incubation normal peritoneal exudate macrophages with rHuEpo (2 IU/ml). Our results showed rHuEpo injection reduced the peritoneal exudate macrophages iron retention. The uptake of Fe(II) was decreased via the suppression of DMT1 (+IRE) expression and the release of Fe(II) was increased with increasing the expression of FPN1 in macrophages. Moreover, the expression of HAMP mRNA was four times lower in rHuEpo-treated liver of rats than control group (CG). HAMP mRNA expression was increased; the synthesis of DMT1 had no significant change, whereas the FPN1 was decreased in normal peritoneal exudate macrophages after treatment with rHuEpo in vitro. We conclude that hepcidin may play a major, causative role in the change of FPN1 synthesis and that decreased the iron retention in macrophages of rHuEpo-treated rats.     

Brain capillary endothelial cells mediate iron transport into the brain by segregating iron from transferrin without the involvement of divalent metal transporter 1

Rats were studied for [(59)Fe-(125)I]transferrin uptake in total brain, and fractions containing brain capillary endothelial cells (BCECs) or neurons and glia. (59)Fe was transported through BCECs, whereas evidence of similar transport of transferrin was questionable. Intravenously injected transferrin localized to BCECs and failed to accumulate within neurons, except near the ventricles. No significant difference in [(125)I]transferrin distribution was observed between Belgrade b/b rats with a mutation in divalent metal transporter I (DMT1), and Belgrade +/b rats with regard to accumulation in vascular and postvascular compartments. (59)Fe occurred in significantly lower amounts in the postvascular compartment in Belgrade b/b rats, indicating impaired iron uptake by transferrin receptor and DMT1-expressing neurons. Immunoprecipitation with transferrin antibodies on brains from Belgrade rats revealed lower uptake of transferrin-bound (59)Fe. In postnatal (P)0 rats, less (59)Fe was transported into the postvascular compartment than at later ages, suggesting that BCECs accumulate iron at P0. Supporting this notion, an in situ perfusion technique revealed that BCECs accumulated ferrous and ferric iron only at P0. However, BCECs at P0 together with those of older age lacked DMT1. In conclusion, BCECs probably mediate iron transport into the brain by segregating iron from transferrin without involvement of DMT1.     

Divalent cation transport by vesicular nucleotide transporter

The vesicular nucleotide transporter (VNUT) is a secretory vesicle protein that is responsible for the vesicular storage and subsequent exocytosis of ATP (Sawada, K., Echigo, N., Juge, N., Miyaji, T., Otsuka, M., Omote, H., and Moriyama, Y. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 5683-5686). Because VNUT actively transports ATP in a membrane potential (Δψ)-dependent manner irrespective of divalent cations such as Mg(2+) and Ca(2+), VNUT recognizes free ATP as a transport substrate. However, whether or not VNUT transports chelating complexes with divalent cations remains unknown. Here, we show that proteoliposomes containing purified VNUT actively took up Mg(2+) when ATP was present, as detected by atomic absorption spectroscopy. The VNUT-containing proteoliposomes also took up radioactive Ca(2+) upon imposing Δψ (positive-inside) but not ΔpH. The Δψ-driven Ca(2+) uptake required ATP and a millimolar concentration of Cl(-), which was inhibited by Evans blue, a specific inhibitor of SLC17-type transporters. VNUT in which Arg-119 was specifically mutated to alanine, the counterpart of the essential amino acid residue of the SLC17 family, lost the ability to take up both ATP and Ca(2+). Ca(2+) uptake was also inhibited in the presence of various divalent cations such as Mg(2+). Kinetic analysis indicated that Ca(2+) or Mg(2+) did not affect the apparent affinity for ATP. RNAi of the VNUT gene in PC12 cells decreased the vesicular Mg(2+) concentration to 67.7%. These results indicate that VNUT transports both nucleotides and divalent cations probably as chelating complexes and suggest that VNUT functions as a divalent cation importer in secretory vesicles under physiological conditions.     

Zip14 is a complex broad-scope metal-ion transporter whose functional properties support roles in the cellular uptake of zinc and nontransferrin-bound iron

Recent studies have shown that overexpression of the transmembrane protein Zrt- and Irt-like protein 14 (Zip14) stimulates the cellular uptake of zinc and nontransferrin-bound iron (NTBI). Here, we directly tested the hypothesis that Zip14 transports free zinc, iron, and other metal ions by using the Xenopus laevis oocyte heterologous expression system, and use of this approach also allowed us to characterize the functional properties of Zip14. Expression of mouse Zip14 in RNA-injected oocytes stimulated the uptake of (55)Fe in the presence of l-ascorbate but not nitrilotriacetic acid, indicating that Zip14 is an iron transporter specific for ferrous ion (Fe(2+)) over ferric ion (Fe(3+)). Zip14-mediated (55)Fe(2+) uptake was saturable (K(0.5) ≈ 2 μM), temperature-dependent (apparent activation energy, E(a) = 15 kcal/mol), pH-sensitive, Ca(2+)-dependent, and inhibited by Co(2+), Mn(2+), and Zn(2+). HCO(3)(-) stimulated (55)Fe(2+) transport. These properties are in close agreement with those of NTBI uptake in the perfused rat liver and in isolated hepatocytes reported in the literature. Zip14 also mediated the uptake of (109)Cd(2+), (54)Mn(2+), and (65)Zn(2+) but not (64)Cu (I or II). (65)Zn(2+) uptake also was saturable (K(0.5) ≈ 2 μM) but, notably, the metal-ion inhibition profile and Ca(2+) dependence of Zn(2+) transport differed from those of Fe(2+) transport, and we propose a model to account for these observations. Our data reveal that Zip14 is a complex, broad-scope metal-ion transporter. Whereas zinc appears to be a preferred substrate under normal conditions, we found that Zip14 is capable of mediating cellular uptake of NTBI characteristic of iron-overload conditions.     

MPTP诱导小鼠黑质区铁摄取和DMT1表达增加

铁在帕金森病(Parkinson‘s disease,PD)的发病机制中起着非常关键的作用,为了探讨PD中铁升高的机制,本实验观察了1-甲基4-苯基—1,2,3,6-四氢吡啶(MPTP)处理小鼠黑质(substantia nigra,SN)内铁摄取及新的铁转运蛋白二价金属离子转运蛋白1(DMT1)的表达变化。结果表明：(1)MPTP处理组小鼠SN内铁染色增高,注射MPTP7d组明显高于3d组。(2)MPTP处理组小鼠,酪氨酸羟化酶(TH)免疫阳性细胞数目显著减少。(3)MPTP处理组小鼠,“-IRE”型DMT1表达在各组中均增加,而“ IRE”型DMT1仅在MPTP处理后7d才出现变化。上述结果提示,这种新发现的哺乳动物跨膜铁转运蛋白表达增加可能是引起MPTP处理小鼠SN中铁升高的关键因素,铁的升高进一步导致DA神经元的死亡。     

DMT 1在THP-1氧化LDL过程中对基因表达的影响

采用RT-PCR法和Western blot法对二价拿属转运蛋白1(divalent metal transporter 1, DMT 1)在人单核细胞系THP-1氧化低密度脂蛋白(low density lipoprotein,LDL)的过程中所起到的作用进行了验证。结果表明,DMT 1参与了THP-1氧化LDL的过程,且DMT 1的表达水平显著升高,为临床心血管疾病的抗氧化治疗提供了新的思路。     

Iron Imports. II. Iron uptake at the apical membrane in the intestine

How does iron enter enterocytes? Ablating SLC11A2, the gene for the divalent metal ion transporter DMT1, supports evidence from the Belgrade rat and mk mouse models establishing DMT1 as the primary mechanism serving apical uptake of nonheme iron. DMT1 harnesses the energy from the proton electrochemical potential gradient to drive active transport of Fe(2+) (and perhaps Mn(2+) and other metal ions) into enterocytes. Fe(III) must first be reduced by ascorbic acid and surface ferrireductases. Among these is duodenal cytochrome B (DcytB), but lack of an obvious phenotype in DcytB (Cybrd1) knockout mice suggests ferrireductase redundancy. Our understanding of heme absorption has lagged, but the time is ripe for gains.     

Functional characterisation and genomic analysis of an epithelial calcium channel (ECaC) from pufferfish, Fugu rubripes

An orthologue to the mammalian epithelial calcium channels, ECaC1 (TRPV5) and ECaC2 (TRPV6), was cloned from gill of pufferfish (Fugu rubripes) and characterised, demonstrating that this gene predates the evolution of land-living vertebrates. The F. rubripes ECaC (FrECaC) protein displays all structural features typical for mammalian ECaCs including three ankyrin repeats, six transmembrane domains, and a putative pore region between TM V and TM VI. Functional expression of FrECaC in Madin-Darby canine kidney (MDCK) cells confirmed that the channel mediates Ca(2+) influx. FrECaC was also permeable to Zn(2+) and, to a small extent, to the Fe(2+) ion. Thus, in addition to a role in Ca(2+) uptake FrECaC might serve as a pathway for zinc and iron acquisition. FrECaC mRNA was highly abundant in the gill, but sparsely present in the intestine. Calcium absorption via FrECaC in pufferfish may be subject to the regulation of 1.25(OH)(2)D(3), estrogen and progesterone as consensus cis regulatory elements for the respective steroid hormone receptors were found in the upstream regulatory region of the FrECaC gene. FrECaC gene organisation is very conserved when compared with mammalian ECaCs. Only one ECaC gene seems to exist in the F. rubripes genome, and the corresponding protein clusters together with ECaC2 from mammals upon phylogenetic analysis. Thus, the two mammalian ECaC genes may originate from a single ancestral ECaC2 gene in vertebrates appearing early in evolution.