Iron uptake and Nramp2/DMT1/DCT1 in human bronchial epithelial cells

The capacity of natural resistance-associated macrophage protein-2 [Nramp2; also called divalent metal transporter-1 (DMT1) and divalent cation transporter-1 (DCT1)] to transport iron and its ubiquitous expression make it a likely candidate for transferrin-independent uptake of iron in peripheral tissues. We tested the hypothesis that non-transferrin-bound iron uptake by airway epithelial cells is associated with Nramp2/DMT1/DCT1 and that exposure to iron can increase Nramp2/DMT1/DCT1 mRNA and protein expression and transport of this metal. Exposure of BEAS-2B cells to ferric ammonium citrate (FAC) resulted in a decrease in Fe(3+) concentration in the supernatant that was dependent on time and initial iron concentration. In the presence of internalized calcein, FAC quenched the fluorescent signal, indicating intracellular transport of the metal. The Nramp2/DMT1/DCT1 mRNA isoform without an iron-response element (IRE) increased with exposure of BEAS-2B cells to FAC. RT-PCR demonstrated no change in the mRNA for the isoform with an IRE. Similarly, Western blot analysis for the isoform without an IRE confirmed an increased expression of this protein after FAC exposure, whereas the isoform with an IRE exhibited no change. Finally, immunohistochemistry revealed an increase in the isoform without an IRE in the rat lung epithelium after instillation of FAC. Comparable to mRNA and protein increases, iron transport was elevated after pretreatment of BEAS-2B cells with iron-containing compounds. We conclude that airway epithelial cells increase mRNA and expression of the Nramp2/DMT1/DCT1 without an IRE after exposure to iron. The increase results in an elevated transport of iron and its probable detoxification by these cells.     

Carboxyl-terminus determinants of the iron transporter DMT1/SLC11A2 isoform II (-IRE/1B) mediate internalization from the plasma membrane into recycling endosomes

Mutations in DMT1 (Nramp2 and Slc11a2) impair iron metabolism and cause microcytic anemia. DMT1 is expressed at the duodenal brush border where it controls uptake of dietary iron and is present at the plasma membrane and in recycling endosomes of most cells, where it is necessary for acquisition of transferrin-associated iron. The goal of this study was to identify signal(s) in the cytoplasmic segments of DMT1 responsible for its subcellular targeting and internalization from the plasma membrane into recycling endosomes. We introduced mutations in the amino terminus (DeltaNT), carboxyl terminus (DeltaCT), as well as in NPAY28-31, YSCF62-65, and YLLNT555-559 motifs of a DMT1 construct bearing an exofacial epitope tag, which allowed labeling of the transporter at the cell surface for kinetic studies. Mutants were stably expressed in LLC-PK1 kidney cells and were studied for transport activity, subcellular localization, cell-surface and recycling pool distribution, and internalization from the plasma membrane. Kinetic studies showed that carboxyl-terminus mutants (DeltaCT and DeltaYLLNT) had an increased fraction of the "recycling pool" that was expressed at the cell surface because of impaired internalization from the plasma membrane. Further cell-surface-labeling and immunofluorescence studies in intact cells showed that the DeltaYLLNT and DeltaCT mutants were targeted to the lysosomal compartment upon internalization. These results suggest that the major signal for internalization and recycling of DMT1 isoform II (-IRE/1B) resides in its carboxyl terminus and that removal of this signal leads to a default lysosomal targeting.     

The significance of the mutated divalent metal transporter (DMT1) on iron transport into the Belgrade rat brain

Brain iron transport and distributional pattern of divalent metal transporter I (DMT1) were studied in homozygous Belgrade rats (b/b) which suffer from a mutation in the DMT1 gene. In adult rats, brain uptake of transferrin-bound iron injected intravenously (i.v.) was significantly lower compared with that in heterozygous Belgrade (+/b) and Wistar rats, whereas transferrin uptake was identical. The difference in iron uptake was not apparent until 30 min after injection. The brain iron concentration was lower, and neuronal transferrin receptor-immunoreactivity higher, in adult b/b rats, thus confirming their iron-deficient stage. Antibodies targeting different sites on the DMT1 molecule consistently detected DMT1 in neurones and choroid plexus at the same level irrespective of strain, but failed to detect DMT1 in brain capillary endothelial cells (BCECs), or macro- or microglial cells. The absence of DMT1 in BCECs was confirmed in immunoblots of purified BCECs. DMT1 was virtually undetectable in neurones of rats aged 18 post-natal days irrespective of strain. Neuronal expression of transferrin receptors and DMT1 in adult rats implies that neurones at this age acquire iron by receptor-mediated endocytosis of transferrin followed by iron transport out of endosomes mediated by DMT1. The existence of the mutated DMT1 molecule in neurones suggests that the low cerebral iron uptake in b/b rats derives from a reduced neuronal uptake rather than an impaired iron transport through the blood-brain barrier.     

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.     

Integrin cytoplasmic domain-associated protein-1 (ICAP-1) interacts with the ROCK-I kinase at the plasma membrane

The integrin cytoplasmic domain-associated protein-1 (ICAP-1) binds via its C-terminal PTB (phosphotyrosine-binding) domain to the cytoplasmic tails of beta1 but not other integrins. Using the yeast two-hybrid assay, we found that ICAP-1 binds the ROCK-I kinase, an effector of the RhoA GTPase. By coimmunoprecipitation we show that ICAP-1 and ROCK form complexes in cells and that ICAP-1 contains two binding sites for ROCK. In cells transfected with both ICAP-1 and ROCK, the proteins colocalized at the cell membrane predominantly in lamellipodia and membrane ruffles, but also in retraction fibers. ROCK was not found at these sites when ICAP-1 was not co-transfected, indicating that ICAP-1 translocated ROCK. In lamellipodia ICAP-1 and ROCK colocalized with endogenous beta1 integrins and this colocalization was also observed with the isolated ICAP-1 PTB domain. The plasma membrane localization of ROCK did not depend on beta1 integrin ligation or ROCK kinase activity, and in truncated ROCK proteins it required the presence of the ICAP-1-binding domain. To show that the interaction was direct, we measured fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (CFP) fused to ICAP-1 and yellow fluorescent protein (YFP) fused to ROCK. FRET was observed in lamellipodia in cells that were induced to spread. These results indicate that ICAP-1-mediated binding of ROCK to beta1 integrin serves to localize the ROCK-I kinase to both the leading edge and the trailing edge where ROCK affects cell migration.     

Dynamic traffic through the recycling compartment couples the metal transporter Nramp2 (DMT1) with the transferrin receptor

Nramp2 (natural resistance-associated macrophage protein 2, also called DMT1 and Slc11a2) is a proton-dependent cation transporter, which plays a central role in iron homeostasis. To study the subcellular distribution and dynamics of the transporter, we generated a construct encoding the long splice variant of Nramp2 (isoform II) tagged with the hemagglutinin epitope on a predicted extracellular loop. Cells stably transfected with this construct revealed the presence of Nramp2 in both the plasma membrane and in an endomembrane compartment. By labeling the exofacial epitope with a pH-sensitive fluorescent indicator, we were able to establish that this variant of Nramp2 resides in a vesicular compartment with an acidic lumen (pH 6.2) and that acidification was maintained by vacuolar-type ATPases. Dual labeling experiments identified this compartment as sorting and recycling endosomes. Kinetic studies by surface labeling with 125I-labeled antibodies established that the fraction of endomembrane Nramp2 was approximately equal to that on the cell surface. The two components are in dynamic equilibrium: surface transporters are internalized continuously via a clathrin and dynamin-dependent process, whereas endosomal Nramp2 is recycled to the plasmalemma by a phosphatidylinositol 3-kinase-dependent exocytic process. Depletion of cholesterol had no discernible effect on Nramp2 internalization, suggesting that rafts or caveolae are not essential. Because the pH at the cell surface and in endosomes differs by >or=1 unit, the rates of transport of Nramp2 at the surface and in endomembrane compartments will differ drastically. Their subcellular colocalization and parallel trafficking suggest that Nramp2 and transferrin receptors are functionally coupled to effect pH-dependent iron uptake across the endosomal membrane.     

Two isoforms of a divalent metal transporter (DMT1) in Schistosoma mansoni suggest a surface-associated pathway for iron absorption in schistosomes

We describe two homologues of the mammalian divalent metal transporter (DMT1) for Schistosoma mansoni, a pathogenic intravascular parasite of humans. Schistosomes have a high nutritional and metabolic demand for iron. Nucleotide sequences of the parasite homologues, designated SmDMT1A and -B, are identical in all but the 5'-regions. The predicted amino acid sequences share at least 60% identity with DMT1 (=Nramp2) of humans, mice, and rats, and at least 55% identity with Nramp1 from mice, humans and Caenorhabditis elegans. SmDMT1A is expressed in differentiating eggs, miracidia, cercariae, schistosomula, and adults, whereas SmDMT1B is expressed in all but the miracidium and occurs at lower levels than SmDMT1A in differentiating eggs and cercariae. An iron-responsive element, present at the 3'-untranslated region of many DMT1 molecules, is not present in schistosome mRNAs studied here. A Western blot analysis of adult worm preparations using a homologous rabbit serum raised against a schistosome DMT1 peptide and a heterologous serum raised against mammalian DMT1, revealed a band approximating 115 kDa. By immunofluorescence microscopy, the schistosome DMT1s localize primarily to the tegument. Iron uptake assays demonstrated that SmDMT1s were able to rescue yeast growth in ferrous iron-transport deficient yeast (fet3fet4). The results suggest that schistosomes express molecules for ferrous iron transport in their tegument, suggesting trans-tegumental transport as one means of iron acquisition for these parasites.     

Protection of cells against membrane damage by haemolytic agents: divalent cations and protons act at the extracellular side of the plasma membrane

The protective effect of Ca2+, Zn2+ and H+ against membrane damage induced by different haemolytic agents has been studied by measuring monovalent cation leakage and haemolysis of erythrocytes, and phosphoryl[3H]choline and adenine nucleotide leakage from Lettre cells prelabelled with [3H]choline. The protective effect of Ca2+ and Zn2+ on erythrocytes damaged by Staphylococcus aureus alpha-toxin, Sendai virus or melittin is unaffected by the addition of A23187, even though this ionophore greatly increases the uptake of 45Ca2+ or 65Zn2+. The same result has been found for the protective effect of Zn2+ on Lettre cells damaged by S. aureus alpha-toxin, Sendai virus, melittin or Triton X-100. Leakage of phosphoryl[3H]choline from prelabelled Lettre cells is inhibited if extracellular pH is lowered; lowering the intracellular pH without affecting the extracellular pH, affords little protection. It is concluded that Ca2+, Zn2+ and H+ protect cells against membrane damage induced by haemolytic agents by an action at the extracellular side of the plasma membrane.     

Rapid regulation of divalent metal transporter (DMT1) protein but not mRNA expression by non-haem iron in human intestinal Caco-2 cells.

A divalent metal transporter, DMT1, located on the apical membrane of intestinal enterocytes is the major pathway for the absorption of dietary non-haem iron. Using human intestinal Caco-2 TC7 cells, we have shown that iron uptake and DMT1 protein in the plasma membrane were significantly decreased by exposure to high iron for 24 h, in a concentration-dependent manner, whereas whole cell DMT1 protein abundance was unaltered. This suggests that part of the response to high iron involved redistribution of DMT1 between the cytosol and cell membrane. These events preceded changes in DMT1 mRNA, which was only decreased following 72 h exposure to high iron.     

Synthesis and biological evaluation of substituted pyrazoles as blockers of divalent metal transporter 1 (DMT1)

Three distinct series of substituted pyrazole blockers of divalent metal transporter 1 (DMT1) were elaborated from the high-throughput screening pyrazolone hit 1. Preliminary hit-to-lead efforts revealed a preference for electron-withdrawing substituents in the 4-amido-5-hydroxypyrazole series 6a-l. In turn, this preference was more pronounced in a series of 4-aryl-5-hydroxypyrazoles 8a-j. The representative analogs 6f and 12f were found to be efficacious in a rodent model of acute iron hyperabsorption. These three series represent promising starting points for lead optimization efforts aimed at the discovery of DMT1 blockers as iron overload therapeutics.     

The natural resistance-associated macrophage protein 1 Slc11a1 (formerly Nramp1) and iron metabolism in macrophages

Slc11a1 (solute carrier family 11 member 1) (formerly Nramp1) modulation of iron metabolism in macrophages plays an important role in early phase macrophage activation, and therefore host innate immunity. This review focuses on the role of Nramp1 in intramacrophage iron metabolism, with emphasis on the two prevailing mechanisms of Nramp1 modulation of iron metabolism in macrophages.     

Uptake of safranine and other lipophilic cations into model membrane systems in response to a membrane potential

Lipophilic cations such as safranine and methyltriphenylphosphonium (MTPP⁺) are commonly employed to obtain measures of the membrane potential (Δψ) exhibited by energized biological membrane systems. These probes reflect the presence of Δψ (inside negative) by accumulating in the interior of the membrane bound system to achieve transmembrane distributions dictated by the Nernst equation. In this work, we characterize the ability of model membrane large unilamellar vesicle systems to accumulate safranine and other biologically active lipophilic cations in response to a K⁺ diffusion potential (interior negative) across the large unilamellar vesicle membrane. We show that safranine, MTPP⁺, chlorpromazine and vinblastine can be rapidly accumulated to achieve interior lipophilic cation concentrations which may be more than two orders of magnitude higher than exterior concentrations. In the case of safranine, for example, incubation of 2 mM safranine with large unilamellar vesicle systems exhibiting a Δψ of −100 mV or more can lead to interior safranine concentrations in excess of 120 mM. This accumulation appears to proceed as an antiport K⁺-safranine exchange process, and the optical ‘safranine response’ observed can be attributed to precipitation of the dye inside the vesicle as the interior concentrations of safranine exceeds its solubility (96 mM). These observations are discussed in terms of the utility of probes such as safranine and MTPP⁺ for determinations of Δψ as well as their implications for the equilibrium transbilayer distributions of biologically active lipophilic cations in vivo.     

Differential microbial clearance and immunoresponse of Balb/c (Nramp1 susceptible) and DBA2 (Nramp1 resistant) mice intracerebrally infected with Mycobacterium bovis BCG (BCG)

In mice, the gene encoding Nramp1 (natural resistance-associated protein 1) exists in two allelic forms, differing for a point mutation. According to Nramp1 genotype, extensive literature documents a clear-cut distinction of inbred strains in two non-overlapping groups that phenotypically express resistance (Nramp1r) and susceptibility (Nramp1s) to systemic infections. Here, we provide evidence that Nramp1r (DBA/2) and Nramp1s (Balb/c) mice differently handle intracerebral infection with Mycobacterium bovis BCG. Distinct trends of microbial clearance from the brain and also different patterns of local immune responses occur, thus arguing on the involvement of Nramp1 gene product on the accomplishment of cerebral anti-mycobacterial defenses.     

Adenosine A2A receptors assemble into higher-order oligomers at the plasma membrane

Oligomerization of G protein-coupled receptors (GPCRs) is known to play important roles in regulating receptor pharmacology and function. Whereas many bivalent GPCR interactions have been described, the stoichiometry and localization of GPCR oligomers are largely unknown. We have used bimolecular fluorescence complementation (BiFC) to study adenosine A_2A receptor (A_2AR) oligomerization. The data suggest specificity of the A_2AR/A_2AR interaction monitored by BiFC and proper sub-cellular localization of tagged receptors. Moreover, using a novel approach combining fluorescence resonance energy transfer and BiFC, we found that at least three A_2A receptors assemble into higher-order oligomers at the plasma membrane in Cath.A differentiated neuronal cells.

Structured summary

MINT-6797156, MINT-6797142: A2AR (uniprotkb:P29274) physically interacts (MI:0218) with A2AR (uniprotkb:P29274) by bimolecular fluorescence complementation (MI:0809)

MINT-6797129: A2AR (uniprotkb:P29274) physically interacts (MI:0218) with A2AR (uniprotkb:P29274) by fluorescent resonance energy transfer (MI:0055)

     

Atomic force microscopy study of the effects of Mg(2+) and other divalent cations on the end-to-end DNA interactions

Atomic force microscopy (AFM) was applied to directly visualize the end-to-end DNA interaction mediated by magnesium cations. We took advantage of the APS-mica, allowing the preparation of samples in a broad range of monovalent and divalent cations to separate the effects of Mg(2+) and Na(+) cations on the interaction of restriction DNA fragments with cohesive end. The AFM data clearly show that DNA restriction fragments with cohesive ends form substantial amount of circles in the presence of Mg(2+) cations, suggesting that Mg(2+) cations stabilize the interaction of cohesive ends. This effect depends on the MgCl(2) concentration, so that the yield of circles approaches 18% in the presence of 50 mM MgCl(2). Furthermore, we demonstrate that this conferred cohesive end stability is specific for divalent cations, as substitution of MgCl(2) with NaCl leads to a near complete loss of cohesive end stability. We further demonstrate that cohesive end stabilization is achieved by substituting Mg(2+) with Ca(2+), Mn(2+), or Zn(2+). The data obtained suggest that the end stabilization mediated by divalent cations is primarily the result of inter-base interactions rather than bridging of phosphate moieties.     

Self-association of des-(B26-B30)-insulin. The effect of Ca2+ and some other divalent cations

It has been confirmed by sedimentation equilibrium and sedimentation velocity experiments that des-(B26-B30)-insulin does not self-associate at neutral pH. Sedimentation equilibrium experiments at pH 7, 25 degrees C were conducted to investigate the effects of the structurally and physiologically important divalent cations Zn2+, Cd2+, Pb2+ and Ca2+ on the aggregation state of des-(B26-B30)-insulin (pig) in solution. It was found that all of these ions bring about association of this insulin analogue; Zn2+ and Cd2+ to a more marked degree than Pb2+ and Ca2+. The predominant species in solutions containing Zn2+ appear to be hexamers and hexameric aggregates, in those containing Cd2+, species up to and including tetramers, and in those containing Pb2+ and Ca2+, monomers and dimers of des-(B26-B30)-insulin appear to be the only species present. The possible significance of these findings, especially in relation to a role for Ca2+ in the action of insulin, is discussed.