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Originally published In Press as doi:10.1074/jbc.M500116200 on January 5, 2005

J. Biol. Chem., Vol. 280, Issue 10, 9635-9639, March 11, 2005
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Stable Plasma Membrane Levels of hCTR1 Mediate Cellular Copper Uptake*

John F. Eisses, Yiqing Chi, and Jack H. Kaplan{ddagger}

From the Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60607

Received for publication, January 4, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The human copper transporter 1 (hCtr1), when heterologously overexpressed in insect cells, mediates saturable Cu uptake. In mammalian expression systems, a rapid Cu-dependent internalization of hCtr1 has been reported in cells that overexpress epitope-tagged hCtr1 when exposed to Cu in the external medium. This finding led to the suggestion that such internalization may be a step in the hCtr1 transmembrane Cu transport mechanism. We have demonstrated that preincubation in Cu-containing media of sf9 cells stably expressing hCtr1 has no effect on the initial rate of Cu transport. Furthermore, Western blot analyses of fractionated sf9 cell membranes show no evidence of a regulatory Cu-dependent internalization from the plasma membrane. In similar studies on human embryonic kidney (HEK) 293 cells, we showed that incubation with Cu does not alter the initial rate of Cu uptake mediated by endogenous levels of hCtr1 compared with untreated cells. Confirmation that hCtr1 mediates this transport is provided by specific small interfering RNA-dependent decreases in hCtr1 protein levels and in Cu transport rates. Western blot analysis and confocal microscopy of human embryonic kidney 293 cells showed that the majority of hCtr1 protein is localized at the plasma membrane and no significant internalization is detected upon Cu treatment. We concluded that internalization of hCtr1 is not a required step in the transport pathway; we suggest that oligomeric hCtr1 acts as a conventional transporter providing a permeation pathway for Cu through the membrane and that internalization of endogenous hCtr1 in response to elevated extracellular Cu levels does not play a significant regulatory role in Cu homeostasis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cu is an essential cofactor for many enzymes in eukaryotic cells, and in recent years there has been an increasing understanding of the homeostatic mechanisms that are used to regulate cellular Cu content and to deliver Cu to its required sites (1, 2). Tightly controlled homeostatic mechanisms are required as Cu is an essential metal; however, as with several other trace metals, excessive metal accumulation is severely toxic (3–5). Cu removal from cells is handled by the ATP7 P-type ATPases, Wilsons and Menkes disease proteins, which utilize ATP hydrolysis to deliver Cu either to the extracellular compartment or into the secretory pathway (1, 6). The protein(s) responsible for Cu uptake has been less well characterized. A major transporter mediating Cu entry into mammalian cells is Ctr1. Ctr1 is functionally related to the Cu uptake systems first identified in yeast about 10 years ago (7, 8). Ctr1 apparently mediates the uptake of Cu(I) into cells (9, 10). It is essential for embryonic development (11, 12), and some progress has been made toward understanding its mechanism, largely through elegant complementation studies in yeast (13).

Utilizing epitope-tagged constructs, it was recently shown that Cu exposure of cells that overexpressed tagged human copper transporter 1 (hCtr1)1 molecules at their surface show a rapid and complete internalization from the plasma membrane (14, 15). This process occurred within 10 min of exposure to Cu levels (5 µM) that were close to the Km of the transporter. It was suggested that this Cu-dependent internalization might be a part of the transport pathway, rather like the internalization of the Fe-bound transferrin receptor in the process of cellular acquisition of iron (14–16). In addition, it was reported that such internalization resulted in degradation of hCtr1 and suggested that the internalization-degradation that was Cu-dependent might be an important regulatory pathway that limited Cu uptake under Cu-replete conditions (14).

hCtr1 consists of 190 amino acid residues. It has an extracellular amino terminus, an intracellular carboxyl terminus, and three transmembrane segments (10, 17). It has been proposed that the methionine-rich amino terminus plays some role in Cu coordination in a functional oligomeric complex (13), but details of the transport mechanism are still the subject of intensive study. During studies on the structure and function of hCtr1 expressed in sf9 insect cells using baculovirus-mediated infection, we had observed that initial rates of Cu uptake remained linear for at least 1 h in the continued presence of extracellular Cu at concentrations ranging from 2 to 25 µM (10). Likewise, others have seen linear uptake for at least 1 h in HEK 293 cells overexpressing epitope-tagged hCtr1 (9). From our studies in insect cells, it seemed unlikely that a large fraction of hCtr1 was rapidly internalized from the plasma membrane. If this had occurred, the initial rate of Cu uptake would have been expected to fall. We have reported here an investigation of this issue in stably transfected insect cells and observed no decrease in the functional activity of hCtr1 following exposure to extracellular Cu. We also extended this approach to endogenous hCtr1 in HEK 293 cells. In this human cell line we do not observe a change in transport activity of endogenous hCtr1 following incubation with Cu or a Cu-dependent internalization from the plasma membrane.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
hCtr1 Detection—An antibody against the carboxyl-terminal 15 amino acids of hCtr1 was raised in rabbits by Affinity BioReagents, Golden, CO. The peptide SWKKAVVVDITEHCH was synthesized and conjugated to KLH. Rabbits were immunized. The resulting antibody was affinity purified using the peptide that it was raised against. Endogenous or overexpressed hCtr1 protein was probed with the hCTR1 antibody at a dilution of 1:50,000.

hCtr1 Expression in Insect Cells—hCtr1 constructs were cloned into pIB/V5-HisTOPO vector (Invitrogen). Wild type hCtr1 and C189S cDNA were PCR amplified from a baculovirus expression vector (10) using primers (5' forward primer, 5'-gaattcatggatcattccc-3', and 3' reverse primer, 5'-ccgcggaacaacttcccactgc-3') that contain EcoRI and SacII restriction sites engineered at the 5'- and 3'-ends, respectively. C189S is a mutant of hCtr1 that has the cysteine at amino acid position 189 mutated to a serine residue and migrates primarily as a monomer, as opposed to multimeric molecules (10). The amplified cDNA constructs were ligated into the pIB/V5-His-TOPO vector. The expression plasmids were sequenced to confirm the correct cDNA sequence for each construct and then were transfected into sf9 cells using Cellfectin transfection reagent according to the manufacturer's protocol (Invitrogen).

sf9 cells containing integrated hCtr1 cDNA were selected using Blasticidin S (Invitrogen) for 2 weeks. Expression of the respective hCtr1 construct was compared with baculovirus-expressed protein and confirmed by Western analysis using our anti-hCtr1 antibody. Cells were maintained in Ex-Cell 420 medium (JRH Biosciences, Lenexa, KS) containing 0.015 mg/ml Blasticidin S. Cu uptake into cells was measured in each of these cell lines using Cu-64, as previously reported (10). A minimum of three determinations of the rate of Cu uptake was performed for each construct or experiment.

RNAi Suppression—Two methods were utilized to generate siRNA molecules to use in RNAi analysis in HEK 293 cells. The first method used oligonucleotides selected and synthesized by Invitrogen (Stealth RNAi). The second method used the Invitrogen Block-iT RNAi kit. Two Stealth RNAi oligonucleotides were tested for knockdown of endogenous hCtr1 protein. The second method involved amplification of hCtr1 and modification of this amplified product using the components of the Block-iT RNAi TOPO transcription kit and the Block-iT Dicer RNAi kit. Briefly, hCtr1 cDNA was amplified using two primers (5'-gaattcatggatcattccc-3' and 5'-aagcttaacaacttcccactgc-3'), and T7 linkers were ligated using the TOPO ligation technology. Equal amounts of sense and antisense single-stranded RNA transcripts were annealed to generate double-stranded RNA complexes that were then cut to create 20–25-bp fragments by the Dicer enzyme. These fragments were transfected into HEK 293 cells and analyzed to determine whether gene knockdown had occurred. Transfection efficiency was monitored using a fluorescent oligonucleotide (BLOCK-iT fluorescent oligo; Invitrogen) and estimated to be 80–90%.

Both sets of RNAi molecules were transfected individually into HEK 293 cells using Lipofectamine 2000 following Invitrogen's protocols. The ability of the RNAi molecules to knock down hCtr1 expression was analyzed by hCtr1 protein detection using anti-hCtr1 antibody on whole cell extracts and by measuring Copper-64 transport in HEK 293 cells transfected with and without RNAi molecules. As a control for nonspecific knockdown, RNAi were generated from a Lac Z cDNA using the Block-iT RNAi kit.

Cell Fractionation—Fractionation of insect cells or HEK 293 cells was carried out using a 5-step sucrose step gradient as described previously (10) or using linear Optiprep gradients (Sigma). Briefly, cells were treated with cycloheximide (100 µg/ml) for 20 min followed by ±100 µM CuCl2 treatment for 2 h. Cells were pelleted by centrifugation, washed twice with PBS, and resuspended in homogenization buffer (0.25 M sucrose, 1 mM EDTA, 10 mM Hepes-NaOH, pH 7.4). Cells were lysed using a dounce homogenizer (20 strokes), and the postnuclear fraction was layered on top of a 5-ml linear gradient. The gradient was centrifuged at 200,000 x g for 3 h and collected in 0.5-ml fractions by tube puncture. 30 µg of protein from each fraction was analyzed by SDS-PAGE. The subsequent protein blot was analyzed using the hCtr1 antibody (1:50,000) and an horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:10,000).

Immunofluorescence—HEK 293 cells were grown in 12-well trays for 24–48 h on sterile glass coverslips. In each experiment, CuCl2 (100 µM) was added to the medium of some wells for 2 h, whereas other wells containing HEK 293 cells had no extra copper added. Cells were fixed and permeabilized by the addition of ice-cold acetone followed by a PBS wash. The cells were blocked in PBS containing 1% bovine serum albumin, 1% gelatin overnight. The cells were probed with a primary antibody at the stated dilution for 1 h followed by PBS washes. The cells were then probed with secondary antibodies (1:2000) for 1 h followed by PBS washes. Samples were mounted using Vectashield with 4',6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, Ca). Immunofluorescence microscopy was performed on a Zeiss LSM510 confocal microscope (Carl Zeiss, Gottingen, Germany).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
hCtr1 Expressed in Insect Cells—We have previously shown that viral expression of hCtr1 in sf9 insect cells provides good yields of functional protein (10). In the present study, we developed and used two stable sf9 cell lines expressing hCtr1. Stable cell lines allow more consistent expression of hCtr1 mutant proteins by removing some of the potential variables associated with a lytic viral expression system. Two cell lines were utilized in this study, one expressing a wild type construct and the second expressing a single cysteine substitution mutant, C189S. We tested the ability of cells expressing these two constructs to transport Cu across the plasma membrane in sf9 cells. The cell line expressing the wild type transporter showed saturable Cu uptake with an apparent Km of 8.2 ± 0.8 µM and a Vmax of 84 ± 3.6 pmol Cu/mg protein/min. Similarly, C189S had an apparent Km of 6.8 ± 0.6 µM and a Vmax of 66 ± 2.3 pmol Cu/mg protein/min (data not shown). The Km values for these two constructs are similar to those reported earlier using the baculovirus-mediated expression system (10).

Uptake experiments were performed using a copper concentration of 5 µM assayed for 65 min. We observed linear uptake of Cu-64 in both cell lines for at least 1 h. Cu-64 uptake was 3.5-fold greater in hCtr1 cell lines than in sf9 cells not expressing hCtr1. There was no demonstrable decrease in the rate of uptake during the 65 min of measurement (Fig. 1).



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  		FIG. 1.
Copper uptake time course in sf9 cells. Copper uptake experiments were carried out on sf9 cells expressing C189S ({blacksquare}) or sf9 cells alone (•) to assess the effect of extracellular Cu. Cells were either treated with 100 µM CuCl2 (closed symbols) for 10 min prior to uptake experiments or left untreated (open symbols). The results shown are typical of those obtained in three independent experiments. Inset, Western analysis of cells expressing C189S (lanes 2 and 4) or sf9 cells alone (lanes 1 and 3). Cells were pretreated with copper (100 µM CuCl2, lanes 3 and 4) or no copper (lanes 1 and 2).

 
Recently, reports suggesting rapid internalization of hCtr1 by extracellular copper have speculated about the role of this regulation on the transport of Cu into cells (14). It has been reported that hCtr1 is internalized at copper levels similar to the apparent Km of hCtr1 for Cu transport. This led to the suggestion that binding of Cu to hCtr1 induces internalization with Cu bound. A subsequent event occurs once the Cu-bound hCtr1 is internalized that allows Cu to be transported from inside the endosome (or some other compartment) to proteins in the cytoplasm of cells, either via hCtr1 or another as yet unidentified protein. It was postulated that this mechanism might provide the basis for Cu uptake into cells (14, 16). This model for Cu transport is reminiscent of transferrin receptor-mediated uptake of iron. We have tested this proposal in insect cells expressing C189S hCtr1 and examined the impact of exposure to extracellular copper on hCtr1 proteins at the plasma membrane. C189S was selected as it is predominantly monomeric in SDS gels (10). Cells stably expressing C189S were pretreated for 10 min with 100 µM CuCl2. The cells were washed with transport buffer, and the rate of uptake into these cells was compared with cells that had not been exposed to Cu. The results are shown in Fig. 1. Clearly the pre-exposure to Cu is without effect on the kinetics of Cu uptake. Internalization of a significant fraction of hCtr1 at these high Cu levels would result in a decrease in the rate of Cu uptake. Similarly, Western blot analysis of C189S cells pretreated or not with 100 µM Cu showed no significant difference in the intensity of the hCtr1 C189S protein in the plasma membrane (Fig. 1, inset). This provides strong evidence that significant internalization of hCtr1 is not a necessary step in the transport mechanism. Consequently, there is no reason to suppose that hCtr1 mediates transport by a mechanism other than a conventional transport pathway.

Endogenous hCTR1 Expression in HEK 293 Cells—Previous studies that have described Cu-dependent internalization have been carried out in mammalian cells that have been engineered to overexpress an epitope-tagged version of hCtr1. We decided to extend our experiments to assess the role that internalization might play in regulation of endogenous hCtr1 levels at the plasma membrane of HEK 293 cells. Utilizing a similar strategy as we described above for insect cells, we measured the rate of isotopic Cu uptake into HEK 293 cells following their incubation in the presence of 50 µM Cu (data not shown), 100 µM Cu, or in the absence of Cu. There was no impact of Cu pretreatment on the uptake of extracellular Cu (Fig. 2) where uptake was linear for at least 80 min following Cu pretreatment, and Western blot analysis of plasma membrane fractions isolated from these two sets of cells showed no significant change in the amount of hCtr1 protein (Fig. 2, inset). To better assess the possibility of shifts of hCtr1 from the plasma membrane to internal locations within the cell, we fractionated cells on a linear gradient after cycloheximide treatment followed by 3 h of copper treatment with 100 µM CuCl2. There was no significant movement of the hCtr1 from the plasma membrane to internal fractions either in cells pretreated with 100 µM CuCl2 or cells receiving no Cu pretreatment (data not shown).



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  		FIG. 2.
Copper pretreatment of HEK 293 cells. HEK 293 cells were pretreated with either 100 µM CuCl2 ({blacksquare}) or no Cu (•) for 10 min prior to uptake experiments. Cells were plated in 12-well culture dishes and allowed to grow to 80–90% confluence. The results shown are typical of results obtained from four independent experiments. Inset,40 µg of total membranes were run on a 10% SDS-PAGE gel and blotted to nitrocellulose membrane. The membrane was probed with anti-hCtr1 (1:50,000). Lanes 1 and 2 represent HEK 293 cells without Cu pretreatment. Lanes 3 and 4 represent HEK 293 cells with Cu pretreatment (100 µg/ml cycloheximide for 20 min, 100 µM CuCl2, 2 h).

 
One question that arises from the experiments with HEK 293 cells is whether or not the major fraction of Cu uptake measured is mediated by endogenous hCtr1 or by some other transporter (see "Discussion"). To examine this issue, we utilized an RNAi approach. We treated HEK 293 cells with specific oligonucleotides designed to reduce hCtr1 protein levels. We examined both the rate of uptake of Cu into cells following RNAi knockdown and the antibody signal reflecting protein levels of hCtr1 in the plasma membrane. There was a significant decrease in hCtr1 expression in cells that have been treated with siRNA compared with control cells (Fig. 3, inset). Similarly, there was an ~80% decrease in the rate of Cu uptake into the treated cells. This confirms that the major fraction of the Cu uptake into HEK 293 cells is mediated by hCtr1 (Fig. 3). These experiments demonstrate that endogenous hCtr1 protein is the primary protein involved in the Cu uptake pathway into these mammalian cells. Significantly, hCtr1 is available to mediate Cu uptake in both replete and Cu-depleted extracellular conditions. We do not see any shift of endogenous hCtr1 protein from the plasma membrane nor do we see any loss of total hCtr1 protein (or significant degradation) from the cellular pools of these cells.



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  		FIG. 3.
RNAi analysis of hCtr1 in HEK 293 cells. siRNA was transfected into HEK 293 cells grown in 12-well culture plates. Copper uptake was measured on HEK 293 cells alone ({blacktriangleup}) or cells transfected with siRNA from control Lac Z cDNA ({diamondsuit}), Stealth hCtr1 siRNA (•), or diced hCtr1 siRNA ({blacksquare}). Results shown are similar to those results obtained in three independent experiments. Inset, Western analysis of HEK 293 cells transfected with RNAi. 30 µg of total membranes were loaded on 10% SDS-PAGE gel from cells used in RNAi uptake experiments (lane 1, HEK 293 cells; lane 2, control siRNA cells; lane 3, diced hCtr1 siRNA cells; lane 4, stealth hCtr1 siRNA cells). Proteins were blotted to nitrocellulose and probed with anti-hCtr1 antibody (1:50,000).

 
Confocal Microscopy of Endogenous hCtr1 Cellular Protein— Recent studies investigating the cellular localization of hCtr1 using confocal microscopy have produced conflicting results. The primary location of hCtr1 protein within the cell has varied depending on which cell line was examined (14, 16). Most of this work has been carried out utilizing overexpressed epitopetagged protein. To complement our characterization of the functional properties of endogenous hCtr1 protein, we utilized confocal microscopy to image the hCtr1 protein pools in HEK 293 cells. Using our hCTR1 antibody (1:500), we probed the cellular location of endogenous hCtr1 in HEK 293 cells. As shown in Fig. 4, we saw a significant amount of antibody staining at the plasma membrane. We next examined the effects that excess Cu in the extracellular medium might have on the amount and location of endogenous hCtr1 protein. HEK 293 cells were plated on coverslips and allowed to grow until 50% confluent. The cells were then either treated with 100 µM CuCl2 or were left in medium containing no excess Cu. The results of these experiments are shown in Fig. 4 (top two panels). We see endogenous hCtr1 protein at the plasma membrane in both Cu-treated cells and in cells with no Cu pretreatment. It is difficult to accurately quantitate the levels of protein found at the plasma membrane, but it is clear that a significant portion remains at the plasma membrane, competent to mediate Cu uptake. To confirm that the hCtr1 staining is at the plasma membrane, we double-labeled HEK 293 cells with our hCTR1 antibody as well as with an antibody raised against the {beta}-subunit of the Na,K ATPase (1:500). The latter protein is not sensitive to extracellular Cu concentration. As can be seen in Fig. 4, G and H, Cu pretreatment has little effect on the localization of either protein and co-localization of these two proteins occurs at the plasma membrane.



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  		FIG. 4.
Localization of endogenous hCtr1 in HEK 293 cells. HEK 293 cells were fixed and permeabilized by incubation in acetone. Cells were probed with primary antibodies directed against hCtr1 (A–D, 1:500 dilution) followed by an Alexa 488 goat anti-rabbit antibody (1:2000 dilution; Molecular Probes, Eugene, Or.). HEK 293 cells were also labeled with TO-PRO-3 iodine (A and B, 1:10,000 dilution; Molecular Probes) or an antibody against the {beta}-subunit of the Na,K ATPase (E and F, 1:500 dilution; Affinity BioReagents) followed by a Cy5 donkey anti-mouse antibody (1:800 dilution, Jackson ImmunoLabs, West Grove, PA.). Panels G and H are a merge of panels C and E (G) and D and F (H) with overlap staining patterns seen as yellow staining. The Cy5 color (blue) has been changed to red to allow easier visualization of the overlap.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The absence of a Cu-dependent decrease in the rate of Cu uptake and no loss of hCtr1 from the plasma membrane of insect cells confirm that internalization of Cu-bound transporter is not an essential step for Cu uptake mediated by hCtr1. It seems likely that hCtr1 mediates Cu uptake in a more conventional manner. We suggest that hCtr1 provides a Cu permeation pathway through the plasma membrane. There have been several studies since the first observations on the function of hCtr1 that have concluded that the 28-kDa protein probably forms oligomers in the plasma membrane (9, 10, 18, 19). It seems likely that a stable oligomer of hCtr1 monomers forms in the cell membrane and that this complex mediates the uptake of Cu into the cell. It has been suggested that these oligomers are probably trimeric, although other structures, tetramers or trimers of dimers, have not been ruled out. We suggest that the important methionine-rich domains in the extracellular amino terminus (10, 13, 17) provide a Cu-coordinating center that localizes Cu to the mouth of the transmembrane transport pathway. hCtr1 remains at the plasma membrane and mediates this metal transport pathway. We are currently investigating the details of this proposal utilizing site-directed mutagenesis and functional transport assays.

The suggestion that elevated extracellular Cu might directly feed back to reduce its own cellular uptake is attractive, especially in the light of post-translational down-regulation of Cu transporters that has been observed in yeast following their internalization (20). The initial report of Cu-dependent internalization of epitope-tagged hCTR1 in HEK 293 cells occurred at Cu levels as low as 2 µM and was essentially complete in 10 min (14).

Subsequent studies support this observation in HEK 293 cells at elevated Cu levels of ~50–100 µM. Although both labeling and confocal microscopic evidence was presented for these epitope-tagged constructs, it should be emphasized that no functional corollary was demonstrated. Studies in other cells (HeLa and Caco2 cells) did not observe such Cu-dependent relocalization of endogenous levels of hCTR1 (16); these authors conclude that these putative regulatory phenomena may be cell-specific. It should be borne in mind that although hCtr1 expression is apparently essential for embryonic development and Cu uptake is its sole known physiological function, it is not the only transporter that can mediate Cu uptake. It has been suggested that in some intestinal cells Cu uptake is mediated by the divalent metal ion transporter, DMT1 (21). In the present work we have shown that in HEK 293 cells, a human cell line, hCTR1, is responsible for at least 80% of the Cu uptake. It is interesting that in intestinal cells it has been reported that Cu uptake is stimulated (and not decreased) by exposure to elevated Cu levels (22, 23). It has yet to be shown which of the Cu uptake proteins mediates the uptake pathway in these intestinal cells.

In the present work we have supplied functional (Cu uptake measurements), biochemical (cell fractionation), RNA knockdown, and confocal microscopic data that together suggest that endogenous levels of hCTR1 are stably expressed in the plasma membrane and remain functional in the face of elevated extracellular Cu. We have suggested that Cu uptake is mediated by hCTR1 by providing a transport pathway across the plasma membrane and that the regulation of cellular Cu content in the face of elevated extracellular Cu levels more likely occurs via regulation of the Cu exit pathways. Such regulation of exit pathways has previously been reported (6) to occur in the relocalization of ATP7A, the Menkes disease protein, from intracellular locations to the plasma membrane in response to elevated Cu levels.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant P01 GM067166, Copper Entry into Human Cells, Project 1 (to J. H. K.). The production of Cu-64 at Washington University School of Medicine is supported by NCI, National Institutes of Health Grant R24 CA86307. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Genetics, University of Illinois at Chicago, 900 S. Ashland Ave., Chicago, IL 60607. Tel.: 312-355-2732; Fax: 312-413-0353; E-mail: kaplanj{at}uic.edu.

1 The abbreviations used are: hCtr1, human copper transporter 1; siRNA, small interfering RNA; RNAi, RNA interference; PBS, phosphate-buffered saline; HEK, human embryonic kidney. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Natalie Barnes for advice on the confocal microscopy work.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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