Divalent Metal Ion Transporter Research Articles

Polymorphisms of the SLC11A1 gene and resistance to bovine tuberculosis in African Zebu cattle

Bovine tuberculosis (BTB) is a considerable health threat to livestock keepers and general communities in many developing countries. Information on genetic resistance or susceptibility because of polymorphisms of candidate genes could be used in making selection decisions for breeding disease tolerant/resistant animals. Here, we investigated associations between polymorphisms at the solute carrier family 11 (proton-coupled divalent metal ion transporters), member 1 gene (SLC11A1, previously known as natural resistant associated macrophage protein 1, NRAMP1), with BTB phenotypes in Chadian cattle. Phenotypes were (i) single intradermal comparative cervical tuberculin test (SICCT) outcome, (ii) presence of gross visible lung lesions, (iii) a bacteriological culture test outcome and (iv) a predicted true BTB infection status using a Bayesian model. All traits were recorded as binary (presence or absence) traits. A total of 211 cattle were genotyped for a microsatellite within the SLC11A1 candidate gene. Standard linear and threshold-liability models regressing BTB traits on copy number of SLC11A1 alleles revealed statistically significant effects of SLC11A1 alleles (P < 0.001) on most BTB traits. Polymorphisms (alleles 211, 215 and 217) are significantly related to lower incidence of BTB traits in Chadian cattle. This is the first study to report the association of SLC11A1 gene polymorphisms with BTB traits in Chadian or any other African cattle breeds.

Folding and assembly of TMD 6‐related segments of DMT 1 in trifluoroethanol aqueous solution

Divalent metal-ion transporter 1 (DMT1) belongs to a large class of metal-ion transporters that drive the translocation of a wide range of divalent metal substrates across membranes toward the cytosol with couple of protons. Two highly conserved histidines in the sixth transmembrane domain (TMD6) are essential for metal transport activity in DMT1. In the present study, we determine the high-resolution structures of three 25-residue peptides, corresponding to TMD6 of the wildtype DMT1 (the segment 255-279) and its H267A and H272A mutants, in 30% TFE-d(2) aqueous solution by the combined use of circular dichroism (CD) and NMR spectroscopies. The wildtype peptide forms an 'α-helix-extended segment-α-helix' structure with two helices spanning over Gly258-Ala262 and Met265-Lys277 linked by a hinge at residues Val263-Ile264. The H267A mutation reduces the hinge to one residue (Ile264), while the H272A mutation extends the flexible region of the central part from Val263 to His267. Diffusion-ordered spectroscopy (DOSY) study demonstrates that all the peptides are self-assembly as trimer in 30% TFE-d(2) aqueous solution. The H272A substitution decreases the intermolecular interaction whereas the H267A substitution may enhance the intermolecular interaction. The specific structure of the discontinuous helix and the self-assembly feature of DMT1-TMD6 may be crucial for its biological function. The changes in conformation and intermolecular interaction induced by histidine substitution may be correlated with the deficiency of DMT1 in metal-ion permeation.

Ndfip1-deficient mice have impaired DMT1 regulation and iron homeostasis

AbstractThe divalent metal ion transporter DMT1 is critical for nonheme iron import. We have previously shown that DMT1 is regulated in vitro by ubiquitination that is facilitated by the adaptor proteins Ndfip1 and Ndfip2. Here we report that in Ndfip1−/− mice fed a low- iron diet, DMT1 expression and activity in duodenal enterocytes are significant higher than in the wild-type animals. This correlates with an increase in serum iron levels and transferrin saturation. Liver and spleen iron stores were also increased in Ndfip1−/− mice fed a normal diet. Counterintuitive to the increase in iron uptake, Ndfip1−/− mice fed a low iron diet develop severe microcytic, hypochromic anemia. We demonstrate that this is due to a combination of iron deficiency and inflammatory disease in Ndfip1−/− mice, because Ndfip1−/−/Rag1−/− immunodeficient mice fed a low iron diet did not develop anemia and showed an iron overload phenotype. These data demonstrate that Ndfip1 is a critical mediator of DMT1 regulation in vivo, particularly under iron restricted conditions.

Structure and metal ion binding of the first transmembrane domain of DMT1

DMT1 is an integral membrane protein with 12 putative transmembrane domains. As a divalent metal ion transporter, it plays an important role in metal ion homeostasis from bacteria to human. Loss-function mutations at the conserved motif DPGN located within the first transmembrane domain (TMD1) of DMT1 indicate the significance of TMD1 in the biological function of the protein. In the present work, we study the structure, topology and metal ion binding of DMT1-TMD1 peptide by nuclear magnetic resonance using sodium dodecyl sulfate and dodecylphosphocholine micelles as membrane mimics. We find that the peptide forms an α-helix-extended segment–α-helix configuration in which the motif DPGN locates at the central flexible region. The N-terminal part of the peptide is deeply embedded in micelles, while the motif section and the C-terminal part are close to the surface of micelles. The peptide can bind to Mn2+ and Co2+ ions by the side chains of the negatively charged residues in the motif section and the C-terminal part of TMD1. The crucial role of the central flexible region and the C-terminal part of TMD1 in metal ion capture is confirmed by the binding of the N-terminal part truncated TMD1 to metal ions.

Hypoxia regulates the ferrous iron uptake and reactive oxygen species level via divalent metal transporter 1 (DMT1) Exon1B by hypoxia‐inducible factor‐1

Hypoxia has been shown to increase the expression of a variety of proteins involved in iron homeostasis, including ceruloplasmin, transferrin, and transferring receptor. Divalent metal transporter 1 (DMT1) is a transmembrane protein that is important in divalent metal ion transport, in particular iron. Although previous studies have provided that DMT1 exon1A is regulated by hypoxia, little is known about the relationship between DMT1 exon1B and hypoxia. Hypoxia-inducible factor 1 (HIF-1) is a transcription factor which is stabilized when mammalian cells are subjected to hypoxia. In this study, we have identified a functional hypoxia-response element (HRE) at position of -327 to -323 (-ACGTG-) in DMT1 exon1B promoter using a combination of bioinformatics and biological approaches. Both the total cellular iron and ferrous uptake increased after hypoxia, decreased after DMT1 RNA interference. Reactive oxygen species (ROS) were also changed by +iron responsive element (IRE) DMT1 exon1B overexpression. These findings implicated DMT1 exon1B was a target gene for HIF-1. Hypoxia might affect cellular iron uptake through regulating the expression of DMT1. When iron was present in excess, cells might be damaged by the ROS production.

Identification of an “α-helix-extended segment-α-helix” conformation of the sixth transmembrane domain in DMT1

DMT1 (divalent metal ion transporter 1) is one member of a family of proton-coupled transporters that facilitate the cellular absorption of divalent metal ions. A pair of mutation-sensitive and highly conserved histidines in the sixth transmembrane domain (TM6) of DMT1 was found to be important for proton-metal ion cotransport. In the present work, we investigate the structures and locations of the peptides from TM6 of DMT1 and its H267A and H272A mutants in SDS micelles by CD and NMR methods. The circular dichroism studies show that the α-helix is a predominant conformation for the wildtype peptide and H267A mutant in SDS micelles, whereas the helicity is evidently decreased for H272A mutant. The pH value has little effect on the α-helical contents of the three peptides. The NMR studies indicate that the wildtype peptide in SDS micelles forms an “α-helix-extended segment-α-helix” structure in which the His267 locates near the central part of the extended segment, while the His272 is involved in the α-helical folding. Both histidines are buried in SDS micelles as evidenced by their p K a values. The structure of the wildtype peptide is evidently changed by the mutations of H267A and H272A. The H267A mutant forms an ordered structure consisting of an α-helix from the C-terminus to the central part and continuous turns in the residual part. The extended structure in the central part of the wildtype peptide is abolished by H267A mutation. The H272A mutation mainly induces unfolding of the short helix in the N-terminal side, while the short helix in the C-terminal side and unordered conformation in the central part remain. All the three peptides are embedded in SDS micelles, and the H267A mutant is inserted more deeply due to increasing hydrophobicity in the central part of the peptide. The specific “α-helix-extended segment-α-helix” structure of TM6 may have an important implication for the binding of the transporter to H + and metal ions and the conformation change induced by the mutations of two highly conserved histidines may be correlated to the deficiency of the transport activity of DMT1.

Interaction of calcium with the human divalent metal-ion transporter-1

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 45Ca 2+ uptake. Instead, we found that Ca 2+ blocked the Fe 2+-evoked currents and inhibited 55Fe 2+ uptake in a noncompetitive manner ( K i ≈ 20 mM). The mechanism of inhibition was independent of voltage and did not involve intracellular Ca 2+ signaling. The alkaline-earth metal ions Ba 2+, Sr 2+, and Mg 2+ also inhibited DMT1-mediated iron-transport activity. We conclude that Ca 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.

Zinc inhibits oxidative stress-induced iron signaling and apoptosis in Caco-2 cells

Studies in humans and animals have suggested negative interactions of iron and zinc during their intestinal absorption. Further, zinc seems to prevent iron-induced oxidative damage in rats, which was hypothesized to be through the modulation of the intracellular iron signaling pathway. The aim of this study was, therefore, to understand the effects of zinc on oxidant-induced iron signaling and cell death in human enterocyte-like Caco-2 cells. We demonstrate that zinc decreases glucose/glucose oxidase (H 2O 2-generating system)-induced iron uptake and inhibits iron-regulatory protein 1 activation and divalent metal ion transporter 1 expression. There was also a concomitant decrease in oxidant-induced intracellular labile iron and restoration of ferritin and metallothionein expression. Further, zinc enhanced the Bcl-2/Bax ratio and reduced caspase-3 activity, leading to inhibition of apoptosis. Interestingly, bathophenanthroline disulfonic acid, an extracellular iron chelator, emulated the effects of zinc except for the reduced ferritin levels. These results suggest that zinc inhibits apoptosis by reducing oxidant-induced iron signaling in Caco-2 cells.

Genetic Effects on Toxic and Essential Elements in Humans: Arsenic, Cadmium, Copper, Lead, Mercury, Selenium, and Zinc in Erythrocytes

Background and objectivesAn excess of toxic trace elements or a deficiency of essential ones has been implicated in many common diseases or public health problems, but little is known about causes of variation between people living within similar environments. We estimated effects of personal and socioeconomic characteristics on concentrations of arsenic (As), cadmium (Cd), copper (Cu), mercury (Hg), lead (Pb), selenium (Se), and zinc (Zn) in erythrocytes and tested for genetic effects using data from twin pairs.MethodsWe used blood samples from 2,926 adult twins living in Australia (1,925 women and 1,001 men; 30–92 years of age) and determined element concentrations in erythrocytes by inductively coupled plasma-mass spectrometry. We assessed associations between element concentrations and personal and socioeconomic characteristics, as well as the sources of genetic and environmental variation and covariation in element concentrations. We evaluated the chromosomal locations of genes affecting these characteristics by linkage analysis in 501 dizygotic twin pairs.ResultsConcentrations of Cu, Se, and Zn, and of As and Hg showed substantial correlations, concentrations of As and Hg due mainly to common genetic effects. Genetic linkage analysis showed significant linkage for Pb [chromosome 3, near SLC4A7 (solute carrier family 4, sodium bicarbonate cotransporter, member 7)] and suggestive linkage for Cd (chromosomes 2, 18, 20, and X), Hg (chromosome 5), Se (chromosomes 4 and 8), and Zn {chromosome 2, near SLC11A1 [solute carrier family 11 (proton-coupled divalent metal ion transporters)]}.ConclusionsAlthough environmental exposure is a precondition for accumulation of toxic elements, individual characteristics and genetic factors are also important. Identification of the contributory genetic polymorphisms will improve our understanding of trace and toxic element uptake and distribution mechanisms.

Genetic iron chelation protects against proteasome inhibition-induced dopamine neuron degeneration

Impairment of the ubiquitin proteasome system (UPS) and iron accumulation in the substantia nigra (SN) have both been implicated in the pathogenesis of Parkinson's disease (PD). We previously reported that chemical iron chelation can protect against proteasome inhibitor lactacystin-induced dopamine (DA) neurodegeneration in vivo. Here, we tested potential neuroprotection via genetic expression of the iron chelator human ferritin heavy chain (H-ferritin). We found that overexpression of H-ferritin in DA neurons significantly reduced lactacystin-induced nigral DA neuron loss and striatal DA depletion. Overexpression of H-ferritin also attenuated elevated levels of total and ferrous iron as well as the divalent metal ion transporter 1 (DMT1) in the SN following lactacystin treatment. In addition, overexpression of H-ferritin alleviated the inhibitory effects of lactacystin on proteasome activity in the nigral tissues. These results suggest that H-ferritin exerts neuroprotection possibly by modulating iron homeostasis and restoring proteasome activity.

Mild steel welding fume causes manganese accumulation and subtle neuroinflammatory changes but not overt neuronal damage in discrete brain regions of rats after short-term inhalation exposure

Serious questions have been raised by occupational health investigators regarding a possible causal association between neurological effects in welders and the presence of manganese (Mn) in welding fume. Male Sprague–Dawley rats were exposed by inhalation to 40 mg/m 3 of gas metal arc-mild steel (MS) welding fume for 3 h/day for 10 days. Generated fume was collected in the animal chamber during exposure, and particle size, composition, and morphology were characterized. At 1 day after the last exposure, metal deposition in different organ systems and neurological responses in dopaminergic brain regions were assessed in exposed animals. The welding particles were composed primarily of a complex of iron (Fe) and Mn and were arranged as chain-like aggregates with a significant number of particles in the nanometer size range. Mn was observed to translocate from the lungs to the kidney and specific brain regions (olfactory bulb, cortex, and cerebellum) after MS fume inhalation. In terms of neurological responses, short-term MS fume inhalation induced significant elevations in divalent metal ion transporter 1 (Dmt1) expression in striatum and midbrain and significant increases in expression of proinflammatory chemokines (Ccl2, Cxcl2) and cytokines (IL1β, TNFα) in striatum. In addition, mRNA and protein expression of glial fibrillary acidic protein (GFAP) was significantly increased in striatum after MS fume exposure. However, the 10-day MS welding fume inhalation did not cause any changes in dopamine and its metabolites or GABA in dopaminergic brain regions nor did it produce overt neural cell damage as assessed by histopathology. In summary, short-term MS welding fume exposure led to translocation of Mn to specific brain regions and induced subtle changes in cell markers of neuroinflammatory and astrogliosis. The neurofunctional significance of these findings currently is being investigated in longer, more chronic welding fume exposure studies.

Rescue of cGMP kinase I and the cause of premature death

Results cGKI-/and the RM have a severe anemia and splenomegalie which is associated with a strongly reduced iron content and expression of the ferritin light chain in the spleen. Furthermore, the mRNA levels of transferrin receptor (TfRc) and divalent metal ion transporter (DMT1) are increased in the spleen. Oral or i.m. administration of iron restores partially the iron levels in the spleen of genetargeted cGKI mice, but affects the anemia only moderately. Examination of the intestinal tract showed a massive ulceration in the duodenum that caused intestinal bleeding in cGKI-/and at the later age in the RM as well.

Mammalian iron transport.

Iron is essential for basic cellular processes but is toxic when present in excess. Consequently, iron transport into and out of cells is tightly regulated. Most iron is delivered to cells bound to plasma transferrin via a process that involves transferrin receptor 1, divalent metal-ion transporter 1 and several other proteins. Non-transferrin-bound iron can also be taken up efficiently by cells, although the mechanism is poorly understood. Cells can divest themselves of iron via the iron export protein ferroportin in conjunction with an iron oxidase. The linking of an oxidoreductase to a membrane permease is a common theme in membrane iron transport. At the systemic level, iron transport is regulated by the liver-derived peptide hepcidin which acts on ferroportin to control iron release to the plasma.

Iron Is Essential for Neuron Development and Memory Function in Mouse Hippocampus

Iron deficiency (ID) is the most prevalent micronutrient deficiency in the world and it affects neurobehavioral outcome. It is unclear whether the effect of dietary ID on the brain is due to the lack of neuronal iron or from other processes occurring in conjunction with ID (e.g. hypoxia due to anemia). We delineated the role of murine Slc11a2 [divalent metal ion transporter-1 (DMT-1)] in hippocampal neuronal iron uptake during development and memory formation. Camk2a gene promoter-driven cre recombinase (Cre) transgene (Camk2a-Cre) mice were mated with Slc11a2 flox/flox mice to obtain nonanemic Slc11a2hipp/hipp (double mutant, hippocampal neuron-specific knockout of Slc11a2hipp/hipp) mice, the first conditionally targeted model of iron uptake in the brain. Slc11a2hipp/hipp mice had lower hippocampal iron content; altered developmental expression of genes involved in iron homeostasis, energy metabolism, and dendrite morphogenesis; reductions in markers for energy metabolism and glutamatergic neurotransmission on magnetic resonance spectroscopy; and altered pyramidal neuron dendrite morphology in area 1 of Ammon’s Horn in the hippocampus. Slc11a2hipp/hipp mice did not reach the criterion on a difficult spatial navigation test but were able to learn a spatial navigation task on an easier version of the Morris water maze (MWM). Learning of the visual cued task did not differ between the Slc11a2WT/WT and Slc11a2hipp/hipp mice. Slc11a2WT/WT mice had upregulation of genes involved in iron uptake and metabolism in response to MWM training, and Slc11a2hipp/hipp mice had differential expression of these genes compared with Slc11a2WT/WT mice. Neuronal iron uptake by DMT-1 is essential for normal hippocampal neuronal development and Slc11a2 expression is induced by spatial memory training. Deletion of Slc11a2 disrupts hippocampal neuronal development and spatial memory behavior.

Hypoxia-inducible factor 2α (EPAS1; HIF2A); solute carrier family 11 (proton-coupled divalent metal ion transporters), member 2 (SLC11A2; DMT1)

Hypoxia-inducible factor 2α (EPAS1; HIF2A); solute carrier family 11 (proton-coupled divalent metal ion transporters), member 2 (SLC11A2; DMT1)

Biophysical Properties of Transmembrane Segment 6 of E.coli MntH Transporter

The Natural Resistance-Associated Macrophage Protein (Nramp) family of secondary active divalent metal ion transporters plays an important role in a variety of biological processes, such as metal ion homeostasis, in virtually all living organisms. Due to its structural and functional homology with eukaryotic Nramps, the E. coli transporter MntH (Proton-dependent Manganese Transporter) represents a prototypic model to advance understanding of structure-function relationship in Nramp family. Synthetic peptides corresponding to the transmembrane (TM) segments of membrane proteins could serve as a suitable alternative model for studying the structure and interaction of the membrane protein TM domains with biological membranes. In this study the synthetic peptide corresponding to the sixth transmembrane segment (TMS6) of E. coli MntH and its two mutants, in which the His211 residue was substituted by arginine or alanine, were used. TMS6 was previously shown to contain two functionally important histidine residues. The H211A mutation preserves bacterial sensitivity to metal ions and facilitate H+ uptake in the presence of metal ions. In contrast, H211R does not induce metal sensitivity (1,2). The secondary structures of TMS6 and its mutants were determined in model membranes and membrane-mimicking organic environments, using CD spectroscopy. The conformation of the peptides exhibited ordered α and β conformations in these milieus. Furthermore, patch clamp measurements demonstrated that TMS6 was able to form multi-state ion channels in the presence of manganese as a physiological substrate of MntH. The mutant H211R does not show any channel-like activity and with the mutant H211A the ion channel activity was rarely observed.1. Lam-Yuk-Tseung, S., Govoni, G., Forbes, J., and Gros, P. (2003) Blood 101, 3699-3707.2. Chaloupka, R., Courville, P., Veyrier, F., Knudsen, B., Tompkins, T.A., and Cellier, M.F. (2005) Biochemistry 44, 726-733.

Iron is a potential key mediator of glutamate excitotoxicity in spinal cord motor neurons

Threohydroxyaspartate (THA)-induced glutamate excitotoxicity in organotypic culture of rat spinal cord is a well-known model of motor neuron degeneration. THA causes accumulation of synaptic glutamate and over stimulation of the postsynaptic receptor by inhibiting glutamate uptake. This model has also been used to identify agents that inhibit glutamate excitotoxicity by increasing the expression of glutamate transporter. We now show that THA also increases iron level in rat spinal cord tissue, with concomitant modulation of key iron transport and storage proteins, including transferrin receptor, divalent metal-ion transporter 1 and ferritin. More significantly, iron chelator deferoxamine (DFO) was able to completely prevent THA-induced motor neuron degeneration. The protective effect of DFO did not involve enhancing glutamate uptake. These data provide new mechanistic insight into THA-induced glutamate excitotoxicity and suggest that blocking THA-induced iron rise alone may be sufficient for prevention of glutamate excitotoxicity.

Regulation of the divalent metal ion transporter DMT1 and iron homeostasis by a ubiquitin-dependent mechanism involving Ndfips and WWP2

AbstractMany ion channels and transporters are regulated by ubiquitination mediated by the Nedd4 family of HECT-type ubiquitin ligases (E3s). These E3s commonly interact with substrates via their WW domains that bind to specific motifs in target proteins. However, not all potential targets of these E3s contain WW-binding motifs. Therefore, accessory proteins may mediate the interaction between Nedd4 family members and their targets. Here we report that the divalent metal ion transporter DMT1, the primary nonheme iron transporter in mammals, is regulated by ubiquitination mediated by the Nedd4 family member WWP2. DMT1 interacts with 2 WW domain-interacting proteins, Ndfip1 and Ndfip2, previously proposed to have roles in protein trafficking. This promotes DMT1 ubiquitination and degradation by WWP2. Consistent with these observations, Ndfip1−/− mice show increased DMT1 activity and a concomitant increase in hepatic iron deposition, indicating an essential function of Ndfip1 in iron homeostasis. This novel mechanism of regulating iron homeostasis suggests that Ndfips and WWP2 may contribute to diseases involving aberrant iron transport.

The third and fourth transmembrane domains of Slc11a1: Comparison of their structures and positioning in phospholipid model membranes

Interactions of two peptides TM3 and TM4, corresponding to the third and fourth transmembrane domains of a divalent metal-ion transporter Slc11a1, respectively, with phospholipid model membranes, including zwitterionic dimyristoylphosphatidylcholine (DMPC) and anionic dimyristoylphosphatidylglycerol (DMPG), are studied in a wide range of peptide-to-lipid (P:L) ratios, and the secondary structures and positioning of the peptides in the lipid bilayers are analyzed, using differential scanning calorimetry (DSC), circular dichroism (CD), and fluorescence techniques. DSC and fluorescence results indicate that both peptides are inserted in the phospholipid bilayers. TM4 is buried in the bilayers more deeply and the insertion position is less affected by pH values, whereas TM3 is less deeply embedded and the position is remarkably shifted toward the direction of the bilayer surface with increasing pH value. The incorporation of TM4 in both lipids causes more perturbation to the packing of the hydrophobic chains of the lipids than TM3. Moreover, the perturbation of TM3 to the lipid packing is pH dependent, whereas the perturbation of TM4 is less pH dependent but with an unusually larger change in the enthalpy of the main transition at pH 5.5 than those at pH 4 and 7. CD data demonstrate that the main discrimination between the secondary structures of TM4 and TM3 is the alpha-helix content, more helicity for TM4 than TM3 in both lipids. The folding of the peptides is also affected by the composition of the phospholipids. Anionic lipid DMPG induces more alpha-helical folding than neutral lipid DMPC for both peptides.

Solute carrier family 11 (proton-coupled divalent metal ion transporters), member 2 (SLC11A2; DMT1)

Solute carrier family 11 (proton-coupled divalent metal ion transporters), member 2 (SLC11A2; DMT1)