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Results for “SLC11A2”

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PubMed2006

Comparison of mammalian cell lines expressing distinct isoforms of divalent metal transporter 1 in a tetracycline-regulated fashion.

DMT1 (divalent metal transporter; also known as SLC11A2, DCT1 or Nramp2) is responsible for ferrous iron uptake in the duodenum, iron exit from endosomes during the transferrin cycle and some transferrin-independent iron uptake in many cells. Four protein isoforms differ by starting in exon 1A or 2 and ending with alternative peptides encoded by mRNA that contains or lacks an IRE (iron responsive element; +/-IRE). We have compared 1A/+IRE and 2/-IRE DMT1 during regulated ectopic expression. HEK-293-F (human embryonic kidney-293-fast growing variant) cells were stably transfected with each construct expressed from a tetracycline-regulated CMV promoter. Reverse transcriptase-PCR analysis showed that construct expression responded to doxycycline. Immunofluorescence staining of cells, using antibodies specific for DMT1 isoforms, confirmed an increase in expression in the plasma membrane and cytosolic vesicles after doxycycline treatment, but with isoform specific distributions. Immunoblotting also revealed stimulation of expression. Nevertheless, both DMT1 isoforms performed similarly in assays for functional properties based on 54Mn2+ and 59Fe2+ uptake. Mn incorporation after doxycycline treatment was approximately 10-fold greater than that of untreated cells, while expression in the untreated cells was approximately 5-fold greater than in the untransfected cells. Uptake of Mn depended on addition of doxycycline, with half maximal response at approximately 1 nM doxycycline. Doxycycline-stimulated Mn and Fe uptake was linear with time for 10 min but not over longer periods. Transport exhibited a pH optimum at approximately 5.5 and dependence on incubation temperature and Mn or Fe concentration. The new cell lines should prove useful for research on metal homoeostasis, toxicological studies and efforts to identify distinctive properties of the isoforms.

Animals↗

PubMed2003

The first external loop of the metal ion transporter DCT1 is involved in metal ion binding and specificity.

The yeast null mutant smf1Delta cannot grow on medium containing EGTA. Expression of Smf1p or the mammalian transporter DCT1 (Slc11a2) suppresses the above-mentioned phenotype. Both can also be expressed in Xenopus oocytes, and the uptake activity and their electrophysiological properties can be studied. We used these systems to analyze the properties of mutations in the predicted external loop I of DCT1. The sensitivity of the transporter to amino acid substitutions in this region is manifested by the mutation G119A, which resulted in almost complete inhibition of the metal ion uptake activity and marked changes in the pre-steady-state currents in Xenopus oocytes. The mutation Q126D abolished the uptake and the electrophysiology, but the double mutant D124A/Q126D partially restored it and changed the metal ion specificity in favor of Fe2+. The maximal pre-steady-state currents at negatively imposed potentials shifted to a lower pH of approximately 5. The triple mutant G119A/D124A/Q126D, which has no apparent transport activity, exhibited remarkable pre-steady-state currents at pH 7.5. Moreover, Zn2+ had a dual effect on this mutant; at pH 7.5 it eliminated the pre-steady state without generating steady-state currents, and at pH 5.5 it induced large pre-steady-state currents. The mutant D124A retained appreciable Fe2+ uptake activity but exhibited very little Mn2+ uptake at pH 5.5 and was abolished at pH 6.5. The properties of the various mutants suggest that loop I is involved in the metal ion binding and its coupling to the proton-driving force.

Amino Acid Sequence↗

PubMed2003

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.

Androstadienes↗

PubMed2006

The molecular circuitry regulating the switch between iron deficiency and overload in mice.

Recent positional cloning of the radiation-induced polycythaemia (Pcm) mutation revealed a 58-bp microdeletion in the promoter region of ferroportin 1 (Fpn1), the sole cellular iron exporter identified to date. Here we report a molecular definition of the regulatory mechanisms governing the dynamic changes in iron balance in Pcm heterozygous mice between 3 and 12 weeks of age. Hepatic and/or duodenal response patterns of iron metabolism genes, such as Trfr, cybrd1, and Slc11a2, explained the transition from early postnatal iron deficiency to iron overload by 12 weeks of age. A significant delay in developmental up-regulation of hepcidin (Hamp), the pivotal hormonal regulator of iron homeostasis, correlated with high levels of Fpn1 expression in hepatic Kupffer cells and duodenal epithelial cells at 7 weeks of age. Conversely, upon up-regulation of Hamp expression at 12 weeks of age, Fpn1 expression decreased, indicative of a Hamp-mediated homeostatic loop. Hamp regulation due to iron did not appear dependent on transcription-level changes of the murine homolog of Hemojuvelin (Rgmc). Aged cohorts of Pcm mice exhibited low levels of Fpn1 expression in the context of an iron-deficient erythropoiesis and profound iron sequestration in reticuloendothelial macrophages, duodenum, and other tissues. Thus, similar to the anemia of chronic disease, these findings demonstrate decreased iron bioavailability due to sustained down-regulation of Fpn1 levels by Hamp. We conclude that regulatory alleles, such as Pcm, with highly dynamic changes in iron balance are ideally suited to interrogate the genetic circuitry regulating iron metabolism.

Animals↗

PubMed2006

Identification of a tyrosine-based motif (YGSI) in the amino terminus of Nramp1 (Slc11a1) that is important for lysosomal targeting.

In macrophages, Nramp1 (Slc11a1) is expressed in lysosomes and restricts replication of intracellular pathogens by removing divalent metals (Mn2+ and Fe2+) from the phagolysosome. Nramp2 (DMT1, Slc11a2) is expressed both at the duodenal brush border where it mediates uptake of dietary iron and ubiquitously at the plasma membrane/recycling endosomes of many cell types where it transports transferrin-associated iron across the endosomal membrane. In Nramp2, a carboxyl-terminal cytoplasmic motif ((555)YLLNT(559)) is critical for internalization and recycling of the transporter from the plasma membrane. Here we studied the subcellular trafficking properties of Nramp1 and investigated the cis-acting sequences responsible for targeting to lysosomes. For this, we constructed and studied Nramp1/Nramp2 chimeric proteins where homologous domains of each protein were exchanged. Chimeras exchanging the amino-(upstream TM1) and carboxyl-terminal (downstream TM12) cytoplasmic segments of both transporters were stably expressed in porcine LLC-PK1 kidney cells and were studied with respect to expression, maturation, stability, cell surface targeting, transport activity, and subcellular localization. An Nramp2 isoform II chimera bearing the amino terminus of Nramp1 was not expressed at the cell surface but was targeted to lysosomes. This lysosomal targeting was abolished by single alanine substitutions at Tyr15 and Ile18 of a (15)YGSI(18) motif present in the amino terminus of Nramp1. These results identify YGSI as a tyrosine-based sorting signal responsible for lysosomal targeting of Nramp1.

Amino Acid Motifs↗

PubMed2006

Recent progress in structure-function analyses of Nramp proton-dependent metal-ion transporters.

The natural resistance-associated macrophage protein (Nramp) homologs form a family of proton-coupled transporters that facilitate the cellular absorption of divalent metal ions (Me2+, including Mn2+, Fe2+, Co2+, and Cd2+). The Nramp, or solute carrier 11 (SLC11), family is conserved in eukaryotes and bacteria. Humans and rodents express 2 parologous genes that are associated with iron disorders and immune diseases. The NRAMP1 (SLC11A1) protein is specific to professional phagocytes and extrudes Me2+ from the phagosome to defend against ingested microbes; polymorphisms in the NRAMP1 gene are associated with various immune diseases. Several isoforms of NRAMP2 (SLC11A2, DMT1, DCT1) are expressed ubiquitously in recycling endosomes or specifically at the apical membrane of epithelial cells in intestine and kidneys, and can contribute to iron overload, whereas mutations impairing NRAMP2 function cause a form of congenital microcytic hypochromic anemia. Structure-function studies, using various experimental models, and mutagenesis approaches have begun to reveal the overall transmembrane organization of Nramp, some of the transmembrane segments (TMS) that are functionally important, and an unusual mechanism coupling Me2+ and proton H+ transport. The approaches used include functional complementation of yeast knockout strains, electrophysiology analyses in Xenopus oocytes, and transport assays that use mammalian and bacterial cells and direct and indirect measurements of SLC11 transporter properties. These complementary studies enabled the identification of TMS1 and 6 as crucial structural segments for Me2+ and H+ symport, and will help develop a deeper understanding of the Nramp transport mechanism and its contribution to Me2+ homeostasis in human health and diseases.

Amino Acid Sequence↗

PubMed2005

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.

Animals↗

PubMed2004

Polymorphisms in xenobiotic metabolism genes and autism.

Autism is a neurodevelopmental syndrome defined by deficits in social reciprocity and communication and by unusual repetitive behaviors. Although there is an underlying genetic predisposition, the etiology of autism is currently unknown. A recent increase in prevalence suggests that genetically determined vulnerability to environmental exposure might contribute to the causation of autism. We performed family-based association studies of polymorphisms in metal-regulatory transcription factor 1(MTF1), a multispecific organic anion transporter (ABCC1), proton-coupled divalent metal ion transporters (SLC11A3 and SLC11A2), paraoxonase 1 (PON1), and glutathione S-transferase (GSTP1) genes in 196 autistic disorder families. There was deviation from the expected pattern of transmission for polymorphisms in MTF1 (Single nucleotide polymorphism database reference identification number, dbSNP rs3790625, P = .02) and divalent metal ion transporter SLC11A3 (dbSNP rs2304704, P = .07) genes. Although these results might represent chance finding, further investigations of genetic variations of metal metabolism in autism are warranted.

Autistic Disorder↗

PubMed2004

Molecular and cellular mechanisms underlying iron transport deficiency in microcytic anemia.

A mutation of the iron transporter Nramp2 (DMT1, Slc11a2) causes microcytic anemia in mk mice and in Belgrade rats by impairing iron absorption in the duodenum and in erythroid cells, causing severe iron deficiency. Both mk and Belgrade animals display a glycine-to-arginine substitution at position 185 (G185R) in the fourth predicted transmembrane domain of Nramp2. To study the molecular basis for the loss of function of Nramp2(G185R), we established cell lines stably expressing extracellularly tagged versions of wild-type (WT) or mutated transporters. Like WT Nramp2, the G185R mutant was able to reach the plasmalemma and endosomal compartments, but with reduced efficiency. Instead, a large fraction of Nramp2(G185R) was detected in the endoplasmic reticulum, where it was unstable and was rapidly degraded by a proteasome-dependent mechanism. Moreover, the stability of the mutant protein that reached the plasma membrane was greatly reduced, further diminishing its surface density at steady state. Last, the specific metal transport activity of plasmalemmal Nramp2(G185R) was found to be significantly depressed, compared with its WT counterpart. Thus, a singlepoint mutation results in multiple biosynthetic and functional defects that combine to produce the impaired iron deficiency that results in microcytic anemia.

Anemia↗