Rate and Regulation of Copper Transport by Human Copper Transporter 1 (hCTR1)

Human copper transporter 1 (hCTR1) is a homotrimer of a 190-amino acid monomer having three transmembrane domains believed to form a pore for copper permeation through the plasma membrane. The hCTR1-mediated copper transport mechanism is not well understood, nor has any measurement been made of the rate at which copper ions are transported by hCTR1. In this study, we estimated the rate of copper transport by the hCTR1 trimer in cultured cells using ⁶⁴Cu uptake assays and quantification of plasma membrane hCTR1. For endogenous hCTR1, we estimated a turnover number of about 10 ions/trimer/s. When overexpressed in HEK293 cells, a second transmembrane domain mutant of hCTR1 (H139R) had a 3-fold higher K_m value and a 4-fold higher turnover number than WT. Truncations of the intracellular C-terminal tail and an AAA substitution of the putative metal-binding HCH C-terminal tripeptide (thought to be required for transport) also exhibited elevated transport rates and K_m values when compared with WT hCTR1. Unlike WT hCTR1, H139R and the C-terminal mutants did not undergo regulatory endocytosis in elevated copper. hCTR1 mutants combining methionine substitutions that block transport (M150L,M154L) on the extracellular side of the pore and the high transport H139R or AAA intracellular side mutations exhibited the blocked transport of M150L,M154L, confirming that Cu⁺ first interacts with the methionines during permeation. Our results show that hCTR1 elements on the intracellular side of the hCTR1 pore, including the carboxyl tail, are not essential for permeation, but serve to regulate the rate of copper entry.     

Copper overload affects copper and iron metabolism in Hep-G2 cells

Divalent metal transporter #1 (DMT1) is responsible for intestinal nonheme Fe apical uptake. However, DMT1 appears to have an additional function in Cu transport in intestinal cells. Because the liver has an essential role in body Cu homeostasis, we examined the potential involvement of Cu in the regulation of DMT1 expression and activity in Hep-G2 cells. Cells exposed to 10 microM Cu exhibited a 22-fold increase in Cu content and a twofold decrease in Fe content compared with cells maintained in 0.4 microM Cu. (64)Cu uptake in Cu-deficient Hep-G2 cells showed a twofold decrease in K(m) compared with cells grown in 10 microM Cu. The decreased K(m) may represent an adaptive response to Cu deficiency. Cells treated with >50 microM Cu, showed an eightfold increase in cytosolic metallothionein. DMT1 protein decreased (35%), suggesting that intracellular Cu caused a reduction of DMT1 protein levels. Our data indicate that, as a result of Cu overload, Hep-G2 cells reduced their Fe content and their DMT1 protein levels. These findings strongly suggest a relationship between Cu and Fe homeostasis in Hep-G2 cells in which Cu accumulation downregulates DMT1 activity.     

An All-Atom Model of the Structure of Human Copper Transporter 1

Human copper transporter 1 (hCTR1) is the major high affinity copper influx transporter in mammalian cells that also mediates uptake of the cancer chemotherapeutic agent cisplatin. A low resolution structure of hCTR1 determined by cryoelectron microscopy was recently published. Several protein structure simulation techniques were used to create an all-atom model of this important transporter using the low resolution structure as a starting point. The all-atom model provides new insights into the roles of specific residues of the N-terminal extracellular domain, the intracellular loop, and C-terminal region in metal ion transport. In particular, the model demonstrates that the central region of the pore contains four sets of methionine triads in the intramembranous region. The structure confirms that two triads of methionine residues delineate the intramembranous region of the transporter, and further identifies two additional methionine triads that are located in the extracellular N-terminal part of the transporter. Together, the four triads create a structure that promotes stepwise transport of metal ions into and then through the intramembranous channel of the transporter via transient thioether bonds to methionine residues. Putative copper-binding sites in the hCTR1 trimer were identified by a program developed by us for prediction of metal-binding sites. These sites correspond well with the known effects of mutations on the ability of the protein to transport copper and cisplatin.     

Copper-dependent Recycling of hCTR1, the Human High Affinity Copper Transporter

Copper is an essential co-factor in many important physiological processes, but at elevated levels it is toxic to cells. Thus at both the organism and cellular level mechanisms have evolved to finely tune copper homeostasis. The protein responsible for copper entry from the circulation in most human cells is hCTR1, a small protein (190 amino acid residues) that functions as a trimer in the plasma membrane. In the present work we employ cell surface biotinylation and isotopic copper uptake studies of overexpressed hCTR1 in HEK293 cells to examine the acute (minutes) response of hCTR1 to changes in extracellular copper. We show that within 10 min of exposure to copper at 2.5 μm or higher, plasma membrane hCTR1 levels are reduced (by ∼40%), with a concomitant reduction in copper uptake rates. We are unable to detect any degradation of internalized hCTR1 in the presence of cycloheximide after up to 2 h of exposure to 0–100 μm copper. Using a reversible biotinylation assay, we quantified internalized hCTR1, which increased upon the addition of copper and corresponded to the hCTR1 lost from the surface. In addition, when extracellular copper is then removed, internalized hCTR1 is promptly (within 30 min) recycled to the plasma membrane. We have shown that in the absence of added extracellular copper, there is a small but detectable amount of internalized hCTR1 that is increased in the presence of copper. Similar studies on endogenous hCTR1 show a cell-specific response to elevated extracellular copper. Copper-dependent internalization and recycling of hCTR1 provides an acute and reversible mechanism for the regulation of cellular copper entry.Copper is an essential micronutrient and plays an important function as a co-factor for a number of cellular processes including oxidative phosphorylation, free radical detoxification, neurotransmitter synthesis, iron metabolism, and maturation of connective tissue (1). Copper in excess of cellular requirements is toxic; therefore cells have developed sophisticated mechanisms for regulating copper acquisition and secretion, thus maintaining a critical copper homeostasis (2, 3). In eukaryotes a family of transporters known as the copper transporter (Ctr) proteins mediate cellular copper uptake (4). Ctr proteins are integral membrane proteins that are structurally conserved with three membrane-spanning domains and a number of methionine rich motifs in the N terminus (5). They contain a sequence of conserved cysteine and histidine residues at or close to the C terminus and are predominantly located at the plasma membrane (6). In the yeast, Saccharomyces cerevisiae, the first high affinity copper transporters, yCtr1 and yCtr3, were identified (7, 8), and this facilitated the identification of the human copper transporter gene, hCTR1,² by functional complementation of yeast high affinity copper uptake mutant, ctr1 (9). The mouse CTR1 is 92% identical to hCTR1 (10), and the deletion of mCTR1 results in early embryonic lethality, suggesting an essential role for the high affinity copper transporter in mammalian growth and development (11).hCTR1 has 190 amino acid residues, three membrane-spanning domains, an extracellular N terminus (of 66 amino acids), a large cytoplasmic loop (of 46 amino acid residues), and a short C-terminal tail (of 15 amino acids) and has been shown to form stable dimers and trimers (12–14). The hCTR1 protein has been shown in ⁶⁴Cu uptake experiments to mediate copper transport with a K_m of 1–5 μm and is thought to transport the reduced form, Cu(I) (12, 13, 15). The extracellular N terminus has both N- and O-linked glycosylation at residues Asn¹⁵ and Thr²⁷, respectively (12, 16, 17), and contains two histidine-rich regions and two methionine motifs that are thought to function in copper binding/sensing. Recent studies showed that mutation or deletion of the methionine residues closest to the first transmembrane domain (Met⁴³ and Met⁴⁵) and the conserved methionine residues in the second transmembrane domain (Met¹⁵⁰ and Met¹⁵⁴) had a large inhibitory effect on ⁶⁴Cu uptake (18, 19). Mutational analysis provided no evidence for the tight binding of copper at any specific residues, and it was proposed that hCTR1 provided a pore for the permeation of copper across the membrane (18). Structural confirmation of such a mechanism was provided in the low resolution structure obtained by cryo-electron microscopy studies on recombinant protein (20, 21).Considerable progress has been made in understanding the biochemical, structure-functional, and molecular aspects of hCTR1-mediated copper transport, although many questions remain unanswered (22). It is also important to determine whether or not hCTR1 has a regulatory role preventing the accumulation of toxic levels of copper and maintaining cellular copper homeostasis. Previous reports on whether or not hCTR1 is involved in an acute response to elevated copper have been somewhat controversial. It has been reported that elevated extracellular copper (1–100 μm) stimulates rapid endocytosis and degradation of hCTR1-Myc-tagged protein in HEK293 cells (23), but also high copper levels had no effect on endogenous hCTR1 localization in both HeLa and Caco-2 cells (14). In a study of overexpressed hCTR1 in insect cells, no evidence was seen of internalization in response to elevated copper (24). Imaging studies have shown that the cellular location of hCTR1 varies among cell lines, CTR1 in MDCK and HEK293 cells resides mainly at the plasma membrane (13, 15, 23, 24). Endogenous hCTR1 is located in cytoplasmic vesicular compartments in HeLa, Caco-2, and HepG2 cell lines with some plasma membrane staining in Caco-2 (14). In intestinal sections, basolateral and subapical staining is seen (15).Previous studies (see above) have utilized internalization of prebound antibody (23) or imaging methods (14) to characterize the response of hCTR1 to elevated copper. In the present work we employed HEK cells overexpressing hCTR1 and used cell surface biotinylation, a sensitive and quantitative measure of CTR1 at the cell surface (15, 17). We have combined this with measurements of hCTR1-mediated ⁶⁴Cu uptake as a functional measure of plasma membrane hCTR1 levels. We find that a fraction (∼40%) of hCTR1 is rapidly internalized in the presence of elevated copper and that there is a concomitant reduction in the hCTR1-mediated copper uptake rate. The internalized transporter is not degraded and can be detected in the cytosol. On removal of extracellular copper, the transporter is recycled promptly to the plasma membrane. Internalization of endogenous CTR1 is also observed in MDCK and HepG2 cells, and no reduction is seen in T47D cells. This is, to our knowledge, the first such report of copper-dependent recycling of hCTR1 in response to copper and represents an acute regulatory mechanism that reversibly modulates cellular copper entry.     

Mouse divalent metal transporter 1 is a copper transporter in HEK293 cells

Divalent Metal Transporter 1 (DMT1) is an apical Fe transporter in the duodenum and is involved in endosomal Fe export. Four protein isoforms have been described for DMT1, two from mRNA with an iron responsive element (IRE) and two from mRNA without it. The sets of two begin in exon 1A or 2. We have characterized copper transport using mouse 2/?IRE DMT1 during regulated ectopic expression. HEK293 cells carrying a TetR:Hyg element were stably transfected with pDEST31 containing a 2/?IRE construct. ⁶⁴Cu¹⁺ incorporation in doxycycline treated cells exhibited 18.6 and 30.0-fold increases in Cu content, respectively when were exposed to 10 and 100 μM of extracellular Cu. Cu content was ~4-fold above that of parent cells or cells carrying just the vector. ⁶⁴Cu uptake in transfected cells pre-incubated with 5 μM of Cu-His revealed a Vmax and Km of 11.98 ± 0.52 pmol mg protein^?1 min^?1 and 2.03 ± 0.03 μM, respectively. Doxycycline-stimulated Cu uptake was linear with time. The rates of apical Cu uptake decreased and transepithelial transport increased when intracellular Cu increased. The optimal pH for Cu transport was 6.5; uptake of Cu was temperature dependent. Silver does not inhibit Cu uptake in cells carrying the vector. In conclusion, Cu uptake in HEK293 cells that over-expressed the 2/?IRE isoform of DMT1 transporter supports our earlier contention that DMT1 transports Cu as Cu¹⁺.     

Influence of Estrogens on Copper Indicators: In Vivo and In Vitro Studies

Classic copper indicators are not sensitive and specific for detecting excess copper exposure when this is higher than customary but not markedly elevated. Serum copper and ceruloplasmin (Cp) are the most commonly used indicators to assess nutritional status of copper. The objective of this paper was to study the influence of estrogens on these indicators and others used to assess early effects of excess copper exposure in humans and the expression of a set of copper related proteins in a hepatic cellular model. For the studies in humans, 107 healthy participants (18–50 years) were allocated as follows: group 1 (n = 39), women assessed on day 7 of their hormonal cycle; group 2 (n = 34), women assessed on day 21 of their hormonal cycle, and group 3 (n = 34, comparison group), healthy men. Participants received 8 mg Cu/day (as copper sulfate) during 6 months. Serum Cp and Cu, Cu–Zn–superoxide dismutase activity, liver function indicators [aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gamma glutamyltransferase (GGT)], and serum Fe and Zn concentrations were measured monthly. In addition, the influence of estradiol on intracellular total copper content, hctr1, dmt1 and shbg mRNA abundance and hCTR1, and DMT1 expression was measured in HepG2 cells. Serum Cu, Fe, and Zn and liver aminotransferases but not Cu–Zn–superoxide dismutase varied depending on sex. Fe nutrition indicators, GGT, and ALT activities showed significant differences between the hormonal phases. Cellular experiments showed that estradiol increased cellular Cu concentration and hCTR1 and DMT1 mRNA expression and changed these proteins expression patterns. Estradiols significantly influence the responses to copper at the whole body and the cellular levels, suggesting that they help maintaining copper availability for metabolic needs.     

Comparison between copper and cisplatin transport mediated by human copper transporter 1 (hCTR1)

Copper transporter 1 (CTR1) is a transmembrane protein that imports copper(i) into yeast and mammalian cells. Surprisingly, the protein also mediates the uptake of platinum anticancer drugs, e.g. cisplatin and carboplatin. To study the effects of several metal-binding residues/motifs of hCTR1 on the transport of both Cu(+) and cisplatin, we have constructed Hela cells that stably express a series of hCTR1 variant proteins including H22-24A, NHA, C189S, hCTR1ΔC, H139R and Y156A, and compared their abilities to regulate the accumulation and cytotoxicity of these metal compounds. Our results demonstrated that the cells expressing the hCTR1 mutants of histidine-rich motifs in the N-terminus (H22-24A, NHA) resulted in a higher basal copper level in the steady state compared to those expressing wild-type protein. However, the cellular accumulation of both copper and cisplatin in these variants was found at a similar level to that of wild type when incubated with an excess of metal compounds (100 μM). The cells expressing hCTR1 variants of H139R and Y156A exhibit lower capacities towards accumulation of copper but not cisplatin. Significantly, cells with the C189S variant partially retained the ability of the wild-type hCTR1 protein to accumulate both copper and cisplatin, while for cells expressing the C-terminus truncated variant of hCTR1 (hCTR1ΔC) this ability was absolutely abolished, suggesting that this motif is crucial for the function of the transporter.     

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.     

DMT1, a physiologically relevant apical Cu1+ transporter of intestinal cells

Despite important advancesin the understanding of copper secretion and excretion, the molecularcomponents of intestinal copper absorption remain a mystery. DMT1, alsoknown as Nramp2 and DCT1, is the transporter responsible for intestinaliron uptake. Electrophysiological evidence suggests that DMT1 can alsobe a copper transporter. Thus we examined the potential role of DMT1 asa copper transporter in intestinal Caco-2 cells. Treatment of cellswith a DMT1 antisense oligonucleotide resulted in 80 and 48%inhibition of iron and copper uptake, respectively. Cells incorporatedconsiderable amounts of copper as Cu¹⁺, whereasCu²⁺ transport was about 10-fold lower. Cu¹⁺inhibited apical Fe²⁺ transport. Fe²⁺, but notFe³⁺, effectively inhibited Cu¹⁺ uptake. Theiron content of the cells influenced both copper and iron uptake. Cellswith low iron content transported fourfold more iron and threefold morecopper than cells with high iron content. These results demonstratethat DMT1 is a physiologically relevant Cu¹⁺ transporter inintestinal cells, indicating that intestinal absorption of copper andiron are intertwined.

     

Characterization of a monoclonal antibody capable of reliably quantifying expression of Human Copper Transporter 1 (hCTR1)

Human copper transporter 1 (hCTR1) is the high-affinity copper influx transporter in mammalian cells that also mediates the influx of cisplatin. Loss of hCTR1 expression has been implicated in the development of resistance to this cancer chemotherapeutic agent. It has turned out to be very difficult to develop antibodies to hCTR1 and polyclonal antibodies produced by different laboratories have yielded conflicting results. We have characterized a newly-available rabbit monoclonal antibody that reacts with an epitope on the N-terminal end of hCTR1 that now permits rigorous identification and quantification of hCTR1 using Western blot analysis. Postnuclear membrane (PNM) preparations made from cells engineered to express high levels of myc-tagged hCTR1, and cells in which the expression of hCTR1 was knocked down, were used to characterize the antibody. The identity of the bands detected was confirmed by immunoprecipitation, surface biotinylation and deglycosylation of myc-tagged hCTR1. Despite the specificity expected of a monoclonal antibody, the anti-hCTR1 detected a variety of bands in whole cell lysates (WCL), which made it difficult to quantify hCTR1. This problem was overcome by isolating post-nuclear membranes and using these for further analysis. Three bands were identified using this antibody in PNM preparations that migrated at 28, 33–35 and 62–64 kDa. Multiple lines of evidence presented here suggest that the 33–35 and 62–64 kDa bands are hCTR1 whereas the 28 kDa band is a cross-reacting protein of unknown identify. The 33–35 kDa band is consistent with the expected MW of the glycosylated hCTR1 monomer. This analysis now permits rigorous identification and quantification of hCTR1.     

Acquisition of dietary copper: a role for anion transporters in intestinal apical copper uptake

Copper is an essential micronutrient in humans and is required for a wide range of physiological processes, including neurotransmitter biosynthesis, oxidative metabolism, protection against reactive oxygen species, and angiogenesis. The first step in the acquisition of dietary copper is absorption from the intestinal lumen. The major human high-affinity copper uptake protein, human copper transporter hCTR1, was recently shown to be at the basolateral or blood side of both intestinal and renal epithelial cell lines and thus does not play a direct role in this initial step. We sought to functionally identify the major transport pathways available for the absorption of dietary copper across the apical intestinal membrane using Caco2 cells, a well-established model for human enterocytes. The initial rate of apical copper uptake into confluent monolayers of Caco2 cells is greatly elevated if amino acids and serum proteins are removed from the growth media. Uptake from buffered saline solutions at neutral pH (but not at lower pH) is inhibited by either d- or l-histidine, unaltered by the removal of sodium ions, and inhibited by ～90% when chloride ions are replaced by gluconate or sulfate. Chloride-dependent copper uptake occurs with Cu(II) or Cu(I), although Cu(I) uptake is not inhibited by histidine, nor by silver ions. A well-characterized inhibitor of anion exchange systems, DIDS, inhibited apical copper uptake by 60-70%, while the addition of Mn(II) or Fe(II), competitive substrates for the divalent metal transporter DMT1, had no effect on copper uptake. We propose that anion exchangers play an unexpected role in copper absorption, utilizing copper-chloride complexes as pseudo-substrates. This pathway is also observed in mouse embryonic fibroblasts, human embryonic kidney cells, and Cos-7 cells. The special environment of low pH, low concentration of protein, and protonation of amino acids in the early intestinal lumen make this pathway especially important in dietary copper acquisition.     

Copper(II) import and reduction are dependent on His-Met clusters in the extracellular amino terminus of human copper transporter-1

Copper(I) is an essential metal for all life forms. Though Cu(II) is the most abundant and stable state, its reduction to Cu(I) via an unclear mechanism is prerequisite for its bioutilization. In eukaryotes, the copper transporter-1 (CTR1) is the primary high-affinity copper importer, although its mechanism and role in Cu(II) reduction remain uncharacterized. Here we show that extracellular amino-terminus of human CTR1 contains two methionine-histidine clusters and neighboring aspartates that distinctly bind Cu(I) and Cu(II) preceding its import. We determined that hCTR1 localizes at the basolateral membrane of polarized MDCK-II cells and that its endocytosis to Common-Recycling-Endosomes is regulated by reduction of Cu(II) to Cu(I) and subsequent Cu(I) coordination by the methionine cluster. We demonstrate the transient binding of both Cu(II) and Cu(I) during the reduction process is facilitated by aspartates that also act as another crucial determinant of hCTR1 endocytosis. Mutating the first Methionine cluster (⁷Met-Gly-Met⁹) and Asp¹³ abrogated copper uptake and endocytosis upon copper treatment. This phenotype could be reverted by treating the cells with reduced and nonreoxidizable Cu(I). We show that histidine clusters, on other hand, bind Cu(II) and are crucial for hCTR1 functioning at limiting copper. Finally, we show that two N-terminal His-Met-Asp clusters exhibit functional complementarity, as the second cluster is sufficient to preserve copper-induced CTR1 endocytosis upon complete deletion of the first cluster. We propose a novel and detailed mechanism by which the two His-Met-Asp residues of hCTR1 amino-terminus not only bind copper, but also maintain its reduced state, crucial for intracellular uptake.     

Copper repletion enhances apical iron uptake and transepithelial iron transport by Caco-2 cells

The influence of copper status on Caco-2 cell apical iron uptake and transepithelial transport was examined. Cells grown for 7-8 days in media supplemented with 1 microM CuCl(2) had 10-fold higher cellular levels of copper compared with control. Copper supplementation did not affect the integrity of differentiated Caco-2 cell monolayers grown on microporous membranes. Copper-repleted cells displayed increased uptake of iron as well as increased transport of iron across the cell monolayer. Northern blot analysis revealed that expression of the apical iron transporter divalent metal transporter-1 (DMT1), the basolateral transporter ferroportin-1 (Fpn1), and the putative ferroxidase hephaestin (Heph) was upregulated by copper supplementation, whereas the recently identified ferrireductase duodenal cytochrome b (Dcytb) was not. These results suggest that DMT1, Fpn1, and Heph are involved in the iron uptake process modulated by copper status. Although a clear role for Dcytb was not identified, an apical surface ferrireductase was modulated by copper status, suggesting that its function also contributes to the enhanced iron uptake by copper-repleted cells. A model is proposed wherein copper promotes iron depletion of intestinal Caco-2 cells, creating a deficiency state that induces upregulation of iron transport factors.     

Human copper transporter hCTR1 mediates basolateral uptake of copper into enterocytes: implications for copper homeostasis

Copper is essential for human growth and survival. Enterocytes mediate the absorption of dietary copper from the intestinal lumen into blood as well as utilizing copper for their biosynthetic needs. Currently, the pathways for copper entry into enterocytes remain poorly understood. We demonstrate that the basolateral copper uptake into intestinal cells greatly exceeds the apical uptake. The basolateral but not apical transport is mediated by the high affinity copper transporter hCTR1. This unanticipated conclusion is supported by cell surface biotinylation and confocal microscopy of endogenous hCTR1 in Caco2 cells as well as copper influx measurements that show saturable high affinity uptake at the basolateral but not the apical membrane. Basolateral localization of hCTR1 and polarized copper uptake are also conserved in T84 cells, models for intestinal crypt cells. The lateral localization of hCTR1 seen in intestinal cell lines is recapitulated in immunohistochemical staining of mouse intestinal sections. Biochemical and functional assays reveal the basolateral localization of hCTR1 also in renal Madin-Darby canine kidney cells and opossum kidney cells. Overexpression of hCTR1 in Madin-Darby canine kidney cells results in both apical and basolateral delivery of the overexpressed protein and greatly enhanced copper uptake at both cell surfaces. We propose a model of intestinal copper uptake in which basolateral hCTR1 plays a key role in the physiologically important delivery of copper from blood to intracellular proteins, whereas its role in the initial apical uptake of dietary copper is indirect.     

Molecular characterization of hCTR1, the human copper uptake protein

We have expressed hCTR1, the human copper transporter, in Sf9 cells using a baculovirus-mediated expression system, and we observed greatly enhanced copper uptake. Western blots showed that the protein is delivered to the plasma membrane, where it mediates saturable copper uptake with a K(m) of approximately 3.5 microm. We also expressed functional transporters where the N-linked glycosylation sites were substituted, and we provided evidence for the extracellular location of the amino terminus. Accessibility of amino-terminal FLAG epitope to antibody prior to permeabilization and of carboxyl-terminal FLAG only after permeabilization confirmed the extracellular location of the amino terminus and established the intracellular location of the carboxyl terminus. Tryptic digestion of hCTR1 occurred within the cytoplasmic loop and generated a 10-Da carboxyl-terminal peptide; cleavage was prevented by the presence of copper. hCTR1 mutants where Cys-161 and Cys-189, the two native cysteines, were replaced with serines also mediated copper uptake, indicating that neither cysteine residue was essential for transport. However, the mutants provided evidence that these residues may stabilize hCTR1 oligomerization. Western blots of hCTR1 in Sf9 cells showed expression levels 100-fold higher than in mammalian (HepG2) cells. The high level of functional expression and the low level of endogenous copper uptake will enable future structure-function analysis of this important protein.     

Gastrointestinal uptake of trace elements are changed during the course of a common human viral (Coxsackievirus B3) infection in mice.

Most infectious diseases are accompanied by a change in levels of several trace elements in the blood. However, it is not known whether changes in the gastrointestinal uptake of trace elements contribute to this event. Coxsackievirus B3 (CVB3), adapted to Balb/c mice, was used to study whether infection induces gene expression of metallothionein (MT1) and divalent-metal transporter 1 (DMT1) in the intestine and liver and hepcidin in the liver, as well as whether trace elements in these tissues are changed accordingly. Quantitative expression of CVB3, MT1, DMT1 and hepcidin was measured by real-time RT-PCR and six trace elements by ICP-MS on days 3, 6 and 9 of the infection. The copper/zinc (Cu/Zn) ratio in serum increased as a response to the infection. High concentrations of virus were found in the intestine and liver on day 3 and in the intestine on day 6. MT1 in the intestine and liver increased on days 3 and 6. The increase of MT1 in the liver correlated positively with Cu and Zn. Hepcidin in the liver showed a non-significant increase on days 3 and 6 of the infection, whereas DMT1 in the intestine decreased on day 9. Accordingly, iron (Fe) in the liver increased progressively during the disease, whereas in the intestine DMT1 was negatively correlated to Fe. Arsenic (As), cadmium (Cd) and mercury (Hg) were found to decrease to various degrees in the intestine, serum and liver. Thus, enteroviral infections, and possibly many other infections, may cause a change in the gastrointestinal uptake of both non-essential and essential trace elements.     

Characterization of the high affinity Zn transporter from Noccaea caerulescens, NcZNT1, and dissection of its promoter for its role in Zn uptake and hyperaccumulation

? In this paper, we conducted a detailed analysis of the ZIP family transporter, NcZNT1, in the zinc (Zn)/cadmium (Cd) hyperaccumulating plant species, Noccaea caerulescens, formerly known as Thlaspi caerulescens. NcZNT1 was previously suggested to be the primary root Zn/Cd uptake transporter. Both a characterization of NcZNT1 transport function in planta and in heterologous systems, and an analysis of NcZNT1 gene expression and NcZNT1 protein localization were carried out. ? We show that NcZNT1 is not only expressed in the root epidermis, but also is highly expressed in the root and shoot vasculature, suggesting a role in long-distance metal transport. Also, NcZNT1 was found to be a plasma membrane transporter that mediates Zn but not Cd, iron (Fe), manganese (Mn) or copper (Cu) uptake into plant cells. ? Two novel regions of the NcZNT1 promoter were identified which may be involved in both the hyperexpression of NcZNT1 and its ability to be regulated by plant Zn status. ? In conclusion, we demonstrate here that NcZNT1 plays a role in Zn and not Cd uptake from the soil, and based on its strong expression in the root and shoot vasculature, could be involved in long-distance transport of Zn from the root to the shoot via the xylem.     

The mechanism of copper uptake mediated by human CTR1: a mutational analysis

Cellular copper uptake is a prerequisite for the biosynthesis of many copper-dependent enzymes; disruption of copper uptake results in embryonic lethality. In humans, copper is transported into cells by hCTR1, a membrane protein, composed of 190 amino acids with only three trans-membrane segments. To provide insight into the mechanism of this unusual transporter, we characterized the functional properties of various hCTR1 mutants stably expressed in Sf9 cells. Most single amino acid substitutions involving charged and potential copper-coordinating residues have some influence on the V(max) and K(m) values for copper uptake but do not greatly alter hCTR1-mediated copper transport. However, there were two notable exceptions. Replacement of Tyr(156) with Ala greatly reduced the maximal transport rate without effect on the K(m) value for copper. Also, replacement of His(139) in the second trans-membrane segment with Arg caused a dramatic increase in the rate of copper uptake and a large increase in the K(m) value for copper. This effect was not seen with an Ala replacement, pointing to the role of a positive charge in modulating copper exit from the pathway. Truncated mutants demonstrated that the deletion of a large portion of the N-terminal domain only slightly decreased the apparent K(m) value for copper and decreased the rate of transport. Similar effects were observed with the removal of the last 11 C-terminal residues. The results suggested that the N and C termini, although nonessential for transport, may have an important role in facilitating the delivery of copper to and retrieving copper from, respectively, the translocation pathway. A model of how hCTR1 mediates copper entry into cells was proposed that included a rate-limiting site in the pore close to the intracellular exit.