Zip14 is a complex broad-scope metal-ion transporter whose

Zip14 is a complex broad-scope metal-ion transporter whosefunctional properties support roles in the cellular uptake of zincand nontransferrin-bound iron

Jorge J. Pinilla-Tenas,1 Brian K. Sparkman,1 Ali Shawki,1 Anthony C. Illing,1 Colin J. Mitchell,1

Ningning Zhao,2 Juan P. Liuzzi,2 Robert J. Cousins,2 Mitchell D. Knutson,2 and Bryan Mackenzie1

1Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio; and2Food Science and Human Nutrition Department, University of Florida, Gainesville, Florida

Submitted 19 November 2010; accepted in final form 6 June 2011

Pinilla-Tenas JJ, Sparkman BK, Shawki A, Illing AC, Mitchell CJ,Zhao N, Liuzzi JP, Cousins RJ, Knutson MD, Mackenzie B. Zip14 is acomplex broad-scope metal-ion transporter whose functional properties sup-port roles in the cellular uptake of zinc and nontransferrin-bound iron. Am JPhysiol Cell Physiol 301: C862–C871, 2011. First published June 8, 2011;doi:10.1152/ajpcell.00479.2010.—Recent studies have shown that overex-pression of the transmembrane protein Zrt- and Irt-like protein 14 (Zip14)stimulates the cellular uptake of zinc and nontransferrin-bound iron (NTBI).Here, we directly tested the hypothesis that Zip14 transports free zinc, iron,and other metal ions by using the Xenopus laevis oocyte heterologousexpression system, and use of this approach also allowed us to characterizethe functional properties of Zip14. Expression of mouse Zip14 in RNA-injected oocytes stimulated the uptake of 55Fe in the presence of L-ascorbatebut not nitrilotriacetic acid, indicating that Zip14 is an iron transporter specificfor ferrous ion (Fe2�) over ferric ion (Fe3�). Zip14-mediated 55Fe2� uptakewas saturable (K0.5 � 2 �M), temperature-dependent (apparent activationenergy, Ea � 15 kcal/mol), pH-sensitive, Ca2�-dependent, and inhibited byCo2�, Mn2�, and Zn2�. HCO3

� stimulated 55Fe2� transport. These proper-ties are in close agreement with those of NTBI uptake in the perfused rat liverand in isolated hepatocytes reported in the literature. Zip14 also mediated theuptake of 109Cd2�, 54Mn2�, and 65Zn2� but not 64Cu (I or II). 65Zn2�

uptake also was saturable (K0.5 � 2 �M) but, notably, the metal-ioninhibition profile and Ca2� dependence of Zn2� transport differed from thoseof Fe2� transport, and we propose a model to account for these observations.Our data reveal that Zip14 is a complex, broad-scope metal-ion transporter.Whereas zinc appears to be a preferred substrate under normal conditions, wefound that Zip14 is capable of mediating cellular uptake of NTBI character-istic of iron-overload conditions.

cadmium transport; hereditary hemochromatosis; iron; homeostasisiron transport; Xenopus laevis oocyte; SLC39A14; thalassemia; zinctransport

IRON-OVERLOAD CONDITIONS (e.g., thalassemia, hereditary hemo-chromatosis) are characterized by the appearance in plasma ofnontransferrin-bound iron (NTBI) and result in cardiomyopa-thy, diabetes, hepatic cancer, and cirrhosis. Identification of theroutes of cellular NTBI uptake will therefore provide noveltargets for therapeutics.

Zrt- and Irt-like protein-14 (Zip14) is a member of a largefamily of mammalian metal-ion transporters, the SLC39 genefamily (6, 11, 12, 22, 26). Zip14 (synonyms SLC39A14,KIAA0062) is strongly expressed in the intestine (25, 35) butits subcellular localization there is not yet clear. Notably, Zip14is abundantly expressed in the liver, heart, and pancreas (25,

35, 42), the major sites of organ damage in iron overload. Ourprevious data identified Zip14 as a candidate route for NTBIuptake since overexpression of Zip14 in human embryonickidney (HEK)293, SF9, or HeLa cell lines stimulated NTBIuptake (14, 25), whereas small interfering RNA (siRNA)suppression of endogenous Zip14 in AML12 mouse hepato-cytes decreased NTBI uptake (25).

We have expressed mouse Zip14 in RNA-injected Xenopusoocytes, an efficient heterologous expression system ideal fordirect assays of membrane transport and tolerant of broadmanipulation of experimental conditions. We used radiotracerassays to test the hypothesis that Zip14 transports free iron andto examine the functional properties and metal-ion substrateprofile of Zip14.

MATERIALS AND METHODS

Reagents. Restriction enzymes were obtained from New EnglandBiolabs (Ipswich, MA). All other reagents were obtained from Sigma-Aldrich (St. Louis, MO) or Research Products International (Prospect,IL) unless otherwise indicated.

Expression of mouse Zip14 and human DMT1 in Xenopus oocytes.We performed laparotomy and ovariectomy on adult female Xeno-pus laevis frogs (Nasco, Fort Atkinson, WI) under 3-aminoethyl-benzoate methanesulfonate anesthesia (0.1% in 1:1 water/ice, byimmersion) following a protocol approved by the University ofCincinnati Institutional Animal Care and Use Committee. Ovariantissue was isolated and treated with collagenase A (Roche Diag-nostics, Indianapolis, IN), and oocytes were isolated and stored at17°C in modified Barths’ medium as previously described (28). Weexpressed in oocytes the Zip14 short isoform transcript, the prod-uct of the mouse SLC39A14 gene, GenBank sequence accessionnumber BC021530, in pCMVSport6 as previously described (25,27, 47) under the SP6 promoter. We linearized the pCMVSport6-Zip14 construct using HpaI and synthesized RNA in vitro using themMESSAGE mMACHINE/SP6 RNA polymerase transcription kit(Applied Biosystems/Ambion, Austin, TX) according to the man-ufacturers’ protocols. Human divalent metal-ion transporter-1(DMT1) isoform 1A/IRE(�) RNA was prepared as described (30,41). Defolliculate stage V-VI oocytes were injected with 50 ng ofRNA and incubated for 3–5 days (Zip14) or 6 days (DMT1) beforebeing used in functional assays.

We also expressed in oocytes enhanced green fluorescence protein(EGFP) fusion proteins of Zip14 and DMT1. To construct NH2-terminal-EGFP-Zip14 (EGFP-Zip14) in pCMVSport6, we used for-ward (5=-CTGCCGCCCCTCACTAGTGCCACCTCC-3=) and re-verse (5=-GGAGGTGGCACTAGTGAGGGGCGGCAG-3=) primersto amplify the EGFP sequence from pEGFP-N1 (Clontech, MountainView, CA) flanked by SpeI restriction sites and ligated the EGFPsequence into the NH2-terminal region of pCMVSport6-Zip14 at anSpeI restriction site we created by site-directed mutagenesis. The

Address for reprint requests and other correspondence: B. Mackenzie, Dept. ofMolecular and Cellular Physiology, Univ. of Cincinnati College of Medicine, POBox 670576, Cincinnati, OH 45267-0576 (e-mail: [email protected]).

Am J Physiol Cell Physiol 301: C862–C871, 2011.First published June 8, 2011; doi:10.1152/ajpcell.00479.2010.

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NH2-terminal-EGFP-Zip14 sequence was then cut out and ligated intothe pOX(�) oocyte expression vector (30) using KpnI and NotI.COOH-terminal-EGFP-tagged DMT1 (DMT1-EGFP), a gift of Dr.Elizabeta Nemeth and Dr. Bo Qiao (David Geffen-UCLA School ofMedicine), was generated by subcloning human DMT1 isoform 1A/IRE(�) cDNA (30) into pEGFP-N3 upstream of the EGFP codingsequence, deleting the intervening 74 nucleotides by site-directedmutagenesis, and subcloning the DMT1-EGFP cDNA back intopOX(�). EGFP-tagged constructs in pOX(�) were linearized usingSnaBI and RNA synthesized as before. Oocytes were injected with 50ng of RNA and incubated 4 (EGFP-Zip14) or 6 days (DMT1-EGFP)before being used for confocal microscopy or immunoblotting ofoocyte membrane fractions.

Analysis of EGFP-Zip14 and DMT1-EGFP fusion-protein expres-sion in oocytes. We imaged EGFP-Zip14 and DMT1-EGFP proteinexpression in the oocyte by using the Zeiss LSM 7 DUO confocallaser-scanning microscope (excitation at 488 nm) fitted with the ECPlan-Neofluar �10/0.3 and LD C-Apochromat �40/1.1 W Korrobjectives to measure emission in the band 500–531 nm at a pinholesetting of 9.9 �m.

Western blot analysis of membrane fractions from oocytes express-ing EGFP-Zip14 or DMT1-EGFP. We separated the total membranefraction from homogenates of oocytes (�20 of each) expressingEGFP-Zip14 or DMT1-EGFP by sucrose-density fractionation asdescribed (5), except that we added protease inhibitor cocktail set I(EMD4Biosciences, Gibbstown, NJ) to all solutions. We used thePierce BCA protein assay kit (Thermo Scientific, Rockford, IL) toestimate protein concentration. Oocyte membrane fractions containing�3 �g of total protein were mixed with Laemmli buffer (1� finalconcentration), heated for 30 min at 37°C, and electrophoreticallyseparated on a sodium dodecylsulfate (SDS)-polyacrylamide gel(7.5% acrylamide). Proteins from the gel were transferred to anOptitran BA-S 85 nitrocellulose blotting membrane (Whatman, Pis-cataway, NJ). The blot was incubated for 1 h in blocking solution [5%nonfat dry milk in Tris-buffered saline (TBS), pH 7.4, containing0.01% Tween 20 (TBST)], followed by a 1-h incubation with a1:5,000 dilution of anti-GFP MAb-2 mouse antibody (Thermo Scien-tific, Rockford, IL). The blot was washed in TBST and then incubatedfor 40 min with a 1:5,000 dilution of ZyMax goat anti-mouse IgGhorseradish peroxidase conjugate (Invitrogen, Carlsbad, CA). Afterwashing in TBST and TBS was completed, immunoreactivity wasvisualized by using SuperSignal West Pico enhanced chemilumines-cent substrate (Thermo Scientific) and X-ray film. We performedreversible Ponceau staining (40) of the blot to obtain an index ofprotein loading in each lane. Signal intensities of the immunoreactivebands and of Ponceau staining were quantified by densitometry byusing GENETOOLS software (SynGene, Frederick, MD).

Media for functional assays in oocytes. Oocytes were superfused orincubated at room temperature (22–25°C), unless otherwise indicated,in a standard transport medium containing 98 mM NaCl, 1 mM KCl,2 mM CaCl2, and 1 mM MgCl2, and buffered using 0–5 mM2-(N-morpholino)ethanesulfonic acid and 0–5 mM N=,N=-diethyl-piperazine (GFS Chemicals, Columbus, OH) to obtain pH 7.5 or asotherwise indicated. Media were supplemented with 1 mM L-ascorbicacid in all experiments with Fe2� (except as indicated in Fig. 3C) andwith other metals as indicated; media were supplemented with 1 mML-histidine in experiments with copper.

We prepared ion-substituted media as follows: 1) a bicarbonate-containing medium was prepared by substituting 30 mM NaCl with 30mM NaHCO3 in transport medium equilibrated for 15 min with 5.6%CO2-94.4% N2 and adjusting final pH immediately after gassing; 2) alow-Cl� medium was prepared by substituting NaCl in standardtransport medium (103 mM Cl�) with sodium isethionate (5 mMCl�); and 3) low-Ca2� or Ca2�-free media were prepared by equimo-lar replacement of CaCl2 with additional MgCl2.

Radiotracer transport assays in oocytes. We used 55Fe (added asFeCl3) at final specific activity of 0.3–1.6 GBq/mg, 45Ca (added as

CaCl2) at final specific activity of 130–620 MBq/mg, and 54Mn(added as MnCl2) at final specific activity of 500–550 MBq/mg, eachobtained from Perkin-Elmer Life Science Products (Boston, MA);109Cd (added as CdCl2) at final specific of activity 57–170 MBq/mgand 65Zn (added as ZnCl2) at final specific activity at 180–420MBq/mg, obtained from the National Laboratory (Oak Ridge, TN);and 64Cu (added as CuCl2) at 1.4–4.9 GBq/mg, obtained fromWashington University-St. Louis (St. Louis, MO).

We determined the time course of 2 �M 55Fe2� accumulationbetween 2 min and 2 h (see Fig. 3A). In subsequent experiments,radiotracer metal-ion uptake was measured over 10 min (with theexception of metal-ion uptake in the presence of bicarbonate, mea-sured over 2 min). We terminated radiotracer uptake by rapidlywashing the oocytes three times in ice-cold pH 7.5 transport mediumcontaining 1 mM L-ascorbic acid. Oocytes were then solubilized using5% (wt/vol) SDS and radiotracer content assayed by liquid scintilla-tion counting using Scintisafe-30% liquid scintillation cocktail (FisherScientific, Pittsburgh, PA).

Concentration-dependence data were fit by a modified Hill function(Eq. 1) for which VS is the velocity (uptake) of substrate S (55Fe2� or65Zn2�), Vmax

S is the derived maximum velocity, S is the concentra-tion of substrate S, K0.5

S the substrate concentration at which velocitywas half-maximal, and nH is the Hill coefficient for S.

VS �Vmax

S · SnH

(K0.5S )nH � SnH

(1)

Uptakes obtained over the range of temperatures �15–30°C werefit by an integrated Arrhenius function (Eq. 2), for which Ea is theapparent activation energy, A is the y-intercept, R is the universal gasconstant (1.987 cal·mol�1·K), T is the absolute temperature, and V isthe velocity (55Fe2� or 65Zn2� uptake).

ln(V) � lnA �Ea

R · T(2)

55Fe2� uptakes obtained over a range of extracellular Ca2� con-centrations of 0.15–6.0 mM were fit by a one-site ligand-bindingfunction (Eq. 3) for which VFe is the velocity (55Fe2� uptake), Vmax

Fe isthe derived maximum velocity, [Ca2�] is the extracellular Ca2�

concentration, and apparent KdCa is the [Ca2�] at which 55Fe2� uptake

velocity was half-maximal.

VFe �Vmax

Fe · [Ca2�]

(KdCa) � [Ca2�]

(3)

Statistical and regression analysis. Statistical and regression anal-yses were performed using SigmaPlot version 11 (Systat Software)with critical significance level � � 0.05. Radiotracer uptake data werepresented as mean and standard deviation (SD) for n independentobservations and analyzed using one-way or two-way ANOVA fol-lowed by pairwise multiple comparisons using the Holm-Šidák test.Data were fit by a linear function or by Eqs. 1–3 by using theleast-squares method of linear or nonlinear regression followed byF-tests of the significance of the fit to the model; SE is the standarderror of the estimate, and P is the significance of the fit. Whereappropriate, fit parameters were compared using Student’s t-test.

RESULTS

EGFP-Zip14 expression in Xenopus oocytes. We used con-focal laser-scanning microscopy to image the expression of anNH2-terminal EGFP-fusion protein of murine Zip14 (EGFP-Zip14) in RNA-injected oocytes (Fig. 1). We observed strongfluorescence throughout most of the plasma membrane but nodetectable intracellular fluorescence. We also examined theexpression of a COOH-terminal EGFP-fusion protein of hu-

C863FUNCTIONAL PROPERTIES OF ZIP14 METAL-ION TRANSPORTER

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man DMT1 isoform 1A/IRE(�) (DMT1-EGFP). DMT1-EGFPwas expressed throughout the plasma membrane, consistentwith the pattern of anti-DMT1 immunofluorescence (sameisoform) described previously (30). We detected no fluores-cence in control oocytes. Whereas the expression pattern ofEGFP-Zip14 was similar to that of DMT1-EGFP, fluorescencewas more intense in oocytes expressing DMT1-EGFP; how-ever, since EGFP fluorescence varies depending on the mi-croenvironment and steric mobility, fluorescence signals forthe two fusion proteins are not directly comparable.

For a semiquantitative comparison of expression levels, weused Western blot analysis (anti-GFP) of membrane fractionsisolated from oocytes expressing EGFP-Zip14 or DMT1-EGFP (Fig. 2). We observed for DMT1-EGFP two strongbands at �80 and �110 kDa. The band at �110 kDa likelyrepresents glycosylated DMT1 (3, 30). For EGFP-Zip14, weobserved three strong bands, at �40, �80, and �170 kDa. Theband at �40 kDa may represent a Zip14 monomer, precursor,or degradation product, whereas the band at �170 kDa likelyrepresents an oligomer. We performed densitometric analysisof immunoreactive bands and used as an index of gel loadingthe densities of reversible Ponceau staining (40) of the blot (not

shown). After normalizing by the amount of protein loaded(which was �25% more for DMT1-EGFP than for Zip14-EGFP), we found that the ratio of protein expression betweenDMT1-EGFP and Zip14-EGFP was 1.7. We considered thepossibility that the 40-kDa Zip14-EGFP band represents anonfunctional peptide, in which case the ratio of proteinexpression is 2.9; however, the 40-kDa band is immunoreac-tive with anti-Zip14 antibody, and we do not expect anysynthesis of free EGFP in the oocyte system, so we suspect the40-kDa band represents a degradation product. In either event,the two proteins are expressed in oocytes on the same order,DMT1-EGFP modestly higher in this preparation (see DISCUS-SION for a comparison of their functional activities).

Zip14 mediates cellular uptake of free iron. We expressedmurine Zip14 in RNA-injected oocytes and used radiotracerassays to characterize its functional properties. In the presenceof L-ascorbic acid, expression of Zip14 stimulated up to 150-fold the uptake of 2 �M 55Fe2� compared with that in controloocytes (Fig. 3). 55Fe2� accumulation was linear from 2 minup to at least 2 h (Fig. 3A). Subsequent transport experimentswere conducted over 10 min, within the linear phase of 55Fe2�

uptake (except see Fig. 6A). Zip14-mediated 55Fe2� uptakewas saturable (Fig. 3B); the Fe2� concentration at which55Fe2� uptake was half-maximal (K0.5

Fe ) was 2.3 0.5 �M. TheHill coefficient (nH

Fe) for Fe2� was �1, indicating a lack ofcooperativity. Whereas Zip14 readily transported Fe2� in thepresence of ascorbate, the uptake of 55Fe added as FeCl3 in thepresence of the Fe(III) chelator nitrilotriacetic acid (NTA) andin the absence of an exogenous reducing agent did not differbetween control oocytes and oocytes expressing Zip14 (Fig.3C). Therefore, Zip14 transports ferrous ion (Fe2�) and notferric ion (Fe3�).

Properties of Zip14-mediated Fe2� transport. Zip14-medi-ated 55Fe2� transport was inhibited by 10-fold excess concen-trations of Cd2�, Co2�, Mn2�, Ni2�, Pb2�, or Zn2� (Fig. 4A).Whereas a 10-fold excess of unlabeled Fe2� inhibited 55Fe2�

uptake less potently (by 57 7% SE) than we expected(�90%) for a simple homogeneous system (44), Cd2�, Co2�,and Zn2� afforded complete inhibition of 55Fe2� transport.

We found that Zip14-mediated 55Fe2� transport was tem-perature dependent in the tested range 16–28°C (Fig. 4B). The

Fig. 1. Imaging of enhanced green fluores-cent protein (EGFP)-Zrt, Irt-like protein14 (Zip14), and divalent metal-ion trans-porter 1 (DMT1)-EGFP expression in Xe-nopus oocytes. Confocal laser-scanningmicroscopy of control oocytes and oocytesexpressing EGFP-Zip14 or DMT1-EGFP.Representative images are presented inwhich the optical slice (7.3 �m at �10magnification or 0.6 �m at �40) approxi-mately bisects the oocyte. Scale bars(white) indicate 0.2 mm.

Fig. 2. Western blot analysis of membrane fractions from Xenopus oocytesexpressing EGFP-Zip14 and DMT1-EGFP by using anti-green fluorescentprotein (GFP) antibody. Each lane (numbered at top) was loaded with mem-brane fractions (�3 �g protein per lane) isolated from the following: lane 1,control oocytes; lanes 2 and 3, oocytes expressing DMT1-EGFP; lanes 4 and5, DMT1; lanes 6 and 7, EGFP-Zip14; lanes 8 and 9, Zip14. Intensities of theimmunoreactive bands in each lane of the Western blot were normalized byquantity of protein loaded in each lane determined by densitometric analysis ofreversible Ponceau staining (40) of the blot (not shown).

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Arrhenius plot was linear over this temperature range and theapparent activation energy (Ea) was 15 2 kcal/mol. Analternative index of temperature dependence, the factor bywhich activity increased with every 10-degree increase in T(Q10), was 2.4 0.3 (obtained from the fit parameters of a3-parameter single-exponential growth function; adjusted r2 �0.91, P � 0.003). Zip14-mediated 55Fe2� transport was pHsensitive (Fig. 4C). Zip14 was active over a narrow pH range,the optimal pH was 7.5, and Zip14-mediated 55Fe2� transportwas abolished at pH 6.0 and below.

Comparison of iron-transport activities mediated by Zip14and DMT1. We compared 55Fe2� transport in oocytes express-ing Zip14 and DMT1. DMT1 is known to be maximallystimulated at low pH (30). The 55Fe2� transport activity at pH5.5 in oocytes expressing DMT1 was 1.6-fold (0.5-fold SE)the 55Fe2� transport activity at pH 7.5 in oocytes expressingZip14 (Fig. 5). We verified that the 55Fe2� transport activitiesof the EGFP-fusion proteins of Zip14 and DMT1 were similarto those of their nontagged counterparts (data not shown).Given the slightly higher protein levels for DMT1 comparedwith Zip14 (Fig. 2), these data indicate that Zip14 and DMT1mediate similar 55Fe2� fluxes per functional unit, i.e., theturnover rates of the transport cycle are similar.

Ion dependence of Zip14-mediated Fe2� transport. Whenwe applied voltage-clamp protocols as described previously(30, 31, 41) in studies of DMT1, we did not observe anymetal-ion-evoked currents or presteady-state currents inoocytes expressing Zip14 (data not shown). We provisionallyconclude that Zip14 is not rheogenic (i.e., net charge move-ment is zero) and considered that Zip14-mediated Fe2� uptakemay be associated with an anion influx or countertransport of(i.e., exchange with) (a) cation(s). Since HCO3

� stimulated zincuptake via human Zip2 expressed in K562 erythroleukemiacells (13), we tested the effects of HCO3

� on Zip14 activity. Wefound that addition of 30 mM HCO3

� to the medium stimulatedby 130% 6% (SE) the uptake of 2 �M 55Fe2� in oocytesexpressing Zip14 (Fig. 6A). In a second preparation, we foundthat excess Zn2� completely inhibited Zip14-mediated 55Fe2�

transport both in the absence and presence of HCO3� (data not

shown, P 0.001); in a third preparation, excess Mn2�

inhibited Zip14-mediated 55Fe2� transport in the absence ofHCO3

� (by 40% 11%, SE) to a similar degree to that in thepresence of HCO3

� (by 65% 5%, SE) (data not shown; P �0.06 for absence cf. presence of HCO3

�). Cl� replacement withthe organic anion isethionate had no effect on Zip14-mediated55Fe2� uptake (Fig. 6B). These data may be interpreted in oneof two ways, such that Zip14-mediated iron-transport activity1) is stimulated by extracellular HCO3

� but is not associatedwith an anion flux; or 2) is associated with a nonspecific anioninflux and that HCO3

� is a preferred anion. In any event, Zip14does not appear to be an obligatory HCO3

� cotransporter(although nominally HCO3

�-free media will contain micromo-lar amounts of HCO3

� arising from atmospheric CO2 alone).Zip14-mediated 55Fe2� transport was dependent on the ex-

tracellular Ca2� concentration (Fig. 6C). 55Fe2� transport washalf-maximal at Ca2� concentration of 0.4 0.1 mM (i.e.,apparent Kd

Ca). In separate experiments (not shown), we foundthat the effect of raising the Ca2� concentration from 0.3 to 2.0mM was to increase nearly fivefold the Imax

Fe for 55Fe2� trans-port (P � 0.018, by Student’s t-test) without effect on K0.5

Fe

(P � 0.96) (data were fit by Eq. 1); i.e., increasing Ca2�

accelerated iron transport without altering the affinity of Zip14for Fe2�.

Metal-ion substrate profile of Zip14. The ZIP (SLC39)family of transporters is known primarily for its role in zinchomeostasis (22), but some ZIP transporters are capable of alsotransporting other metal ions. We therefore examined thesubstrate profile of Zip14 by direct measurement of radiotraceruptake. As well as stimulating the uptake of 55Fe2�, expressionof Zip14 in oocytes increased the uptake of 2 �M 109Cd2�,54Mn2�, and 65Zn2� (Fig. 7A); of these, the flux of 55Fe2� wasthe greatest and 54Mn2� the lowest. Zip14 expression did notalter the rate of 64Cu2� uptake either in the presence or absenceof L-ascorbic acid compared with control oocytes (Fig. 7B).Therefore, Zip14 is capable of transporting Fe2�, Cd2�, Mn2�,and Zn2� but not Cu� or Cu2�.

Properties of Zip14-mediated Zn2� transport. The uptake of65Zn2� in oocytes expressing Zip14 was saturable (Fig. 8A);

Fig. 3. Zip14 mediates cellular uptake of free iron. A: time course of uptake of 2 �M 55Fe2� (in the presence of 1 mM L-ascorbic acid) in control oocytes andoocytes expressing Zip14 (n � 8–11 per group). Linear regression of the data for Zip14 (black symbols and lines) yielded a slope of 0.89 0.02 pmol/min andy-intercept at �1.6 0.9 pmol (adjusted r2 � 1.0; P 0.001). For control (gray symbols and lines), the regression had slope 0.005 0.0002 pmol/minand y-intercept 0.02 0.01 pmol (adjusted r2 � 0.99; P 0.001). B: 55Fe2� saturation kinetics in the range 0.2–50 �M Fe2� in the presence of 1 mM L-ascorbicacid (n � 14–16). Data for Zip14 (black symbols and lines) were fit by Eq. 1 yielding parameters Vmax

Fe � 1.1 0.1 pmol/min, nHFe � 1.1 0.2, K0.5

Fe �2.3 0.5 �M (adjusted r2 � 0.97, P 0.001). 55Fe2� uptake was measured at 0.2 and 50 �M Fe2� in control oocytes (gray symbols and lines) and joinedby a linear fit. C: uptake of 2 �M 55Fe from media containing 1 mM nitrilotriacetic acid (NTA) (in place of L-ascorbic acid, L-Asc) or 1 mM L-ascorbic acidin control oocytes and oocytes expressing Zip14 (n � 11–13). Two-way ANOVA revealed an interaction (P 0.001); within NTA, Zip14 did not differ fromcontrol (unadjusted P � 0.90).



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the Zn2� concentration at which 65Zn2� uptake was half-maximal (K0.5

Zn) was 1.9 0.6 �M and was not significantlydifferent from the K0.5

Fe we obtained for 55Fe2� transport (Fig.3B) (P � 0.66, by Student’s t-test). Zip14-mediated uptake of 2�M 65Zn2� was strongly inhibited by 10-fold excess unlabeled Zn2�

(by 88% 3% SE) and more weakly by Cd2� (56% 3%) but wasonly marginally inhibited by Fe2� (14% 5%) and not by any othermetal ion tested (Fig. 8B). Therefore, the metal-ion inhibition profilefor 65Zn2� transport differed from that for 55Fe2� transport.

We found that Zip14-mediated 65Zn2� transport was tem-perature dependent in the tested range 15–30°C (Fig. 8C). TheArrhenius plot was linear over this temperature range; theapparent Ea was 14 2 kcal/mol and was not significantlydifferent from that obtained for 55Fe2� transport (Fig. 4B) (P �0.65, by Student’s t-test). HCO3

� stimulated 65Zn2� transportin oocytes expressing Zip14 (data not shown, P 0.001) in thesame manner as it did for 55Fe2� transport (Fig. 6A). Zip14-mediated 65Zn2� transport was pH sensitive (Fig. 8D). The

optimal pH for 65Zn2� transport was 7.5 (as for 55Fe2� trans-port, Fig. 4C); however, 65Zn2� transport activity was ob-served over a broader pH range than was 55Fe2� transport, and65Zn2� transport activity persisted at pH 6.0 or lower. Zip14-mediated transport of 2 �M 65Zn2� was not affected byreplacement of extracellular Ca2� by Mg2� (Fig. 8E). There-fore, whereas Zip14 transported Fe2� and Zn2� with identicalK0.5 and temperature-dependence parameters, the properties ofZip14-mediated 65Zn2� transport differed from those of 55Fe2�

transport with respect to the metal-ion inhibition profile, pHdependence, and Ca2� dependence.

Metal-ion inhibition profiles for Zip14-mediated Cd2� andMn2� transport. Uptake of 2 �M 109Cd2� was inhibited by10-fold excess unlabeled Cd2� or Zn2� but not by any othermetal ion tested (Fig. 9A). In contrast, the uptake of 2 �M54Mn2� was inhibited by a broad range of divalent metal ionsat 10-fold excess, including Cd2�, Co2�, Fe2�, Ni2�, Pb2�,and Zn2� (Fig. 9B). Therefore the metal-ion inhibition profilefor Zip14-mediated Cd2� transport resembles that for Zn2�

transport (Fig. 8B), whereas the metal-ion inhibition profile forZip14-mediated Mn2� transport resembles that for Fe2� trans-port (Fig. 4A). Likewise, the pH ranges of Zip14-mediatedCd2� and Mn2� transport activities resembled those of Zn2�

and Fe2� transport activities, respectively (data not shown).HCO3

� stimulated Zip14-mediated 109Cd2� transport (interac-tion P � 0.040) and 54Mn2� transport (interaction P 0.001)(data not shown) in the same manner as it did for 55Fe2�

transport (Fig. 6A); however, whereas HCO3� markedly stim-

ulated transport of Fe2� and Mn2�, HCO3� only modestly

stimulated transport of Zn2� and Cd2�.Zip14-mediated Ca2� transport and the effects of divalent

metal ions. Since we found Zip14-mediated 55Fe2� transport tobe Ca2� dependent, we examined whether Zip14 could medi-ate the influx of 45Ca2� (at physiological concentrations) eitherin the presence or absence of divalent metal ions. To reducebackground activity, we included in the media 100 �M niflu-mic acid since we found (in experiments not shown) that doingso reduced by 86% 20% (SE) the endogenous uptake of 150�M 45Ca2� in control oocytes. Expression of Zip14 stimulatedthe uptake of 45Ca2� at 150 or 300 �M in the absence ofdivalent transition metal ions (Fig. 10, A and B). Notably, the45Ca2� fluxes at these concentrations were lower than the fluxeswe had obtained for 55Fe2� or 65Zn2� transport at �1/100th theconcentration (Figs. 3 and 8). 45Ca2� transport was inhibited by

Fig. 5. Comparison of the iron-transport activities of mouse Zip14 and humanDMT1. Uptake of 2 �M 55Fe2� was measured at pH 5.5 and 7.5 in controloocytes (gray bars) and oocytes expressing mouse Zip14 (black bars) or humanDMT1 isoform 1A/IRE(�) (hatched bars) (n � 9–12). ANOVA, P 0.001;aUnadjusted P � 0.90 cf. control at pH 5.5; bunadjusted P � 0.52 cf. controlat pH 7.5; DMT1 at pH 5.5 differed from Zip14 at pH 7.5 (unadjusted P 0.001).

Fig. 4. Properties of Zip14-mediated Fe2� transport. A: metal-ion inhibitionprofile of Zip14-mediated 55Fe2� transport. Uptake of 2 �M 55Fe2� in theabsence (None) or presence of a range of candidate inhibitor metal ions eachat 20 �M, in the presence of 1 mM L-ascorbic acid, in control oocytes (graybars) and oocytes expressing Zip14 (black bars) (n � 10–14). Within Zip14,all metals inhibited 55Fe2� uptake (P 0.001). B: temperature (T) dependenceof Zip14-mediated uptake of 2 �M 55Fe2� (n � 9–13). Data werefit by Eq. 2 to obtain activation energy (Ea) � 15.2 2.0 kcal/mol, ln(A) �25.0 3.5 (adjusted r2 � 0.90, P 0.001). For clarity, control data are notdisplayed. C: uptake of 2 �M 55Fe2� as a function of extracellular pH inoocytes expressing Zip14 (black symbols and line, n � 27–30). Within Zip14,uptakes at each pH differed from one another (unadjusted P 0.007) exceptpH 6.5 cf. pH 8.5 (unadjusted P � 0.20), pH 7.5 cf. pH 8.0 (unadjusted P �0.44), and pH 6.0 cf. pH 5.5 (unadjusted P � 0.54). For this experiment,uptakes in control oocytes (gray symbols and line) were tested only at pH 5.5and 8.5 (n � 31–32). Zip14 did not differ from control at pH 5.5 (unadjustedP � 0.77) but did at pH 8.5 (unadjusted P � 0.004).



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lower concentrations of unlabeled Cd2� (30 �M) or Zn2� (15�M) but not by Fe2� (30 �M) (Fig. 10, A and B).

DISCUSSION

Functional properties of Zip14 support a role in the cellularuptake of nontransferrin-bound iron. We have examined thefunctional properties of mouse Zip14 expressed in RNA-injected Xenopus oocytes and found that Zip14 is capable oftransporting free iron. Zip14 is specific for ferrous ion (Fe2�),whereas ferric ion (Fe3�) is not transported. It is generallyconsidered that most NTBI is present in plasma as Fe(III)ci-trate; however, a significant portion of the iron at the plasmamembrane is expected to be reduced since 1) plasma L-ascorbic

acid concentrations are typically �70 �M, 2) mammalianhepatocytes (34) and other cell types (19, 20) express surfaceferrireductases, and 3) citrate is capable of forming chelates ofboth Fe(II) and Fe(III).

Zip14-mediated 55Fe2� uptake was saturable (K0.5Fe � 2 �M),

temperature-dependent (apparent activation energy, Ea � 15kcal/mol), Ca2� dependent (Kd

Ca � 0.4 mM), and inhibited byCo2�, Mn2�, and Zn2�. HCO3

� stimulated 55Fe2� transport.These data agree well with those obtained from measurementsof NTBI uptake in other preparations. For example, NTBIuptake in the perfused rat liver was temperature dependent(Ea � 14 kcal/mol), Ca2� dependent (Kd

Ca � 0.6 mM), andinhibited by Co2�, Mn2�, and Zn2 (46). The K0.5

Fe obtained inthe study just cited was 16 �M, higher than the K0.5

Fe weobtained in oocytes expressing mouse Zip14; however, higherK0.5 estimates are expected in perfusion studies since bindingby other membrane proteins cannot easily be controlled. In asecond study, iron uptake in isolated rat hepatocytes wasmediated by a high-affinity transport system (K0.5

Fe � 1.3 �M)that was Ca2� dependent (Kd

Ca of 0.6–0.75 mM); however,inhibition by other divalent metal ions was not observed in thatpreparation (2).

Substantial increases in plasma NTBI are characteristic ofiron-overload disorders (e.g., thalassemia, hereditary hemo-chromatosis) (1, 8, 16, 18, 37). Consistent with the functionalproperties of Zip14, a role for Zip14 in NTBI uptake in vivo issupported by the tissue distribution of Zip14 and its subcellularlocalization. Zip14 is a plasma-membrane protein (25) that isabundantly expressed in the liver, heart, and pancreas (35, 42),the three organs that preferentially accumulate iron during ironoverload.

Zip14 is a complex, broad-scope metal-ion transporter.Zip14 also mediated the uptake of 109Cd2�, 54Mn2�, and65Zn2� but not 64Cu (I or II). Zip14-mediated 65Zn2� uptake inoocytes also was saturable (K0.5

Zn �2 �M) and displayed kineticproperties reminiscent of zinc uptake in cultured rat hepato-cytes (38). Notably, the properties of Zn2� transport in oocytesexpressing Zip14 differed from those of Fe2� transport.109Cd2� and 65Zn2� transport escaped inhibition by all metalsexcept those two (Fe2� afforded only weak inhibition of65Zn2� transport), whereas 55Fe2� and 54Mn2� transport wasinhibited by every divalent metal ion tested. This observationreminds us of the importance of determining the substrateprofile of transporters by directly measuring transport of eachcandidate substrate instead of relying on inferences from theinhibition profile for just one radiolabeled test substrate. At

Fig. 6. Ion dependence of Zip14-mediated 55Fe2�

transport. A: effect of bicarbonate (HCO3�) on

Zip14-mediated uptake of 2 �M 55Fe2� (n � 10–11) measured over 2 min to minimize pH changeswith time; two-way ANOVA revealed an interaction(P 0.001). B: effect of Cl� replacement withisethionate (Iseth) on Zip14-mediated uptake of 2�M 55Fe2� (n � 12–15); two-way ANOVA re-vealed no effect of Cl� replacement (P � 0.58) andno interaction (P � 0.58). C: uptake of 2 �M 55Fe2�

as a function of extracellular calcium concentration([Ca2�]o) in oocytes expressing Zip14 (black, n �9–11); data were fit by Eq. 3 to obtain Vmax

Fe � 1.0 0.1 pmol/min, Kd

Ca � 0.39 0.12 mM (adjustedr2 � 0.90, P � 0.002). Data for control oocytes(gray, n � 10) were joined by a linear fit.

Fig. 7. Metal-ion substrate profile of Zip14. A: uptake of radionuclide metalions (*Me2�, each at 2 �M in the presence of 1 mM L-ascorbic acid) in controloocytes and oocytes expressing Zip14 (n � 17–27). Two-way ANOVArevealed an interaction (P 0.001); for all metals, Zip14 differed from control(unadjusted P 0.001); within Zip14, all metals differed from one another(109Cd2� vs. 65Zn2�, unadjusted P � 0.039; all other pairwise comparisons,unadjusted P 0.001). B: uptake of 2 �M 64Cu was measured in the presenceof 1 mM L-histidine and in the presence (64Cu1�) or absence (64Cu2�) of 1 mML-ascorbic acid and compared with uptake of 2 �M 55Fe2� in the presence of1 mM L-ascorbic acid in control oocytes and oocytes expressing Zip14 (n �13–15). Two-way ANOVA revealed an interaction (P 0.001); Zip14 did notdiffer from control for 64Cu1� (P � 1.0) or 64Cu2� (P � 0.56) but differed for55Fe2� (P 0.001).



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least one group has relied on the latter approach and interpretedthe lack of inhibition of Zip14-mediated 109Cd2� uptake byFe2� in viral-infected mouse fetal fibroblasts (15) to contradictour earlier observation that Zip14 could transport iron (25).Given the likelihood that Zip14 shares basic mechanistic prop-erties in common with other members of the mammalianSLC39 family of Zip transporters, the substrate profiles oftransporters within the family may not be fully represented atpresent. For example, the conclusions that mouse Zip1, Zip2,and human ZIP4 and ZIP5 are zinc-specific transporters basedon the lack of inhibition by other metals (9, 10, 45) may needto be reexamined.

The pattern of incomplete mutual inhibition observed herebetween the divalent metal ions tested is not consistent with asingle saturable transport process (44). Whereas Zip14-medi-ated 55Fe2� transport was completely inhibited by excessZn2�, we found that Fe2� only marginally inhibited 65Zn2�

transport. We found that 55Fe2� transport was Ca2� dependent,whereas 65Zn2� transport was not, and that Cd2� and Zn2�, butnot Fe2�, inhibited the modest 45Ca2� fluxes observed inoocytes expressing Zip14. A model that can explain these

observations is one in which there exist two metal-ion trans-location pathways within Zip14. Fe2� transport via the first ofthese translocation pathways is dependent on (i.e., functionallycoupled with) Ca2� transport via the second. Mutual inhibitionof Fe2� and Mn2� transport activities indicates that thesemetals share the first translocation pathway. Zn2� and Cd2�

however are capable of coordinating with both pathways,thereby completely inhibiting Fe2� transport both by compet-ing for the first translocation pathway and by inhibiting theCa2� transport otherwise supporting Fe2� transport. The addi-tional ionic species that account(s) for the apparent net neutraltransport activity of Zip14 are (is) not presently understood.

We provisionally conclude that Zip14 is not rheogenic (i.e.,net charge movement is zero) based on the lack of anymetal-ion-evoked currents in oocytes expressing Zip14. Whilethe ratio of protein expression of DMT1-EGFP relative toZip14-EGFP (see RESULTS and Fig. 2) was 1.7 (or 2.9 if the�40-kDa band for Zip14-EGFP is excluded), the ratio of theirfunctional activities (Fig. 5) was 1.6. Therefore, the two pro-teins are equally active (1.7/1.6 � 1) or, at most, Zip14-EGFPis 1.8 times (2.9/1.6) as active per functional unit (assuming

Fig. 8. Properties of 65Zn2� transport. A: 65Zn2� saturation kinetics in the range 0.1–10 �M Zn2� (n � 10–11). Data for Zip14 (black symbols and line) werefit by Eq. 1 yielding parameters Vmax

Zn � 1.8 0.2 pmol/min, nHZn � 0.9 0.1, K0.5

Zn � 1.9 0.6 �M (adjusted r2 � 0.99, P 0.001). 65Zn2� uptake was measuredat 0.1, 1.0, and 10 �M Zn2� in control oocytes (gray symbols and line) and fit by linear regression. B: metal-ion inhibition profile of Zip14-mediated 65Zn2�

transport. Uptake of 2 �M 65Zn2� in the absence (None) or presence of a range of candidate inhibitor metal ions each at 20 �M, in the presence of 1 mML-ascorbic acid, in control oocytes (gray bars), and oocytes expressing Zip14 (black bars) (n � 10–14). Within Zip14, each metal ion inhibited 65Zn2� uptake(aunadjusted P 0.001, cunadjusted P � 0.003) except b,dnot significant (bunadjusted P � 0.87, dunadjusted P � 0.13). C: temperature dependence ofZip14-mediated uptake of 2 �M 65Zn2� (n � 10–11). Data were fit by Eq. 2 to obtain Ea � 13.9 1.8 kcal/mol, ln(A) � 24.0 3.1 (adjusted r2 � 0.92,P � 0.002). For clarity, control data are not displayed. D: uptake of 2 �M 65Zn2� as a function of extracellular pH in oocytes expressing Zip14 (black symbolsand line, n � 9–13). Within Zip14, uptakes did not differ from one another within the pH ranges marked by the bars above the graph (not significant, unadjustedP � 0.006) except for pH 6.5 cf. pH 7.5 (unadjusted P 0.001); all other pairwise comparisons, unadjusted P 0.001. Uptakes in control oocytes (gray symbolsand line) were tested only at pH 5.5 and 8.5 (n � 11–13). Zip14 did not differ from control at pH 5.5 (unadjusted P � 0.17) but did at pH 8.5 (unadjustedP 0.001). E: uptake of 2 �M 65Zn2� in the presence of 2 mM Ca2� (black bars) or its absence (hatched bars) in control oocytes and oocytes expressing Zip14(n � 32–35). Two-way ANOVA revealed a lack of interaction (P � 0.46).



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that each operates as a monomer). Whereas DMT1 exhibitslarge Fe2�-evoked currents (30), Zip14 does not. Fluxes of upto 2.5 pmol/min observed in nonclamped Zip14-expressingoocytes in this study should correspond to a pure divalentmetal-ion current of �8 nA (converted using the Faraday) overan order of magnitude greater than the noise in the voltageclamp (�0.5 nA root mean square), and much larger currentswould be expected under voltage clamp at �70 mV. In a recentstudy (24), investigators reconstituted a bacterial homologZIPB into liposomes and observed a nonsaturable, voltage-dependent channel-like activity for ZIPB. However, bacterialZIPB shares with mouse Zip14 only 15% identity at the aminoacid level (pairwise alignment using VectorNTI software, In-vitrogen, Carlsbad, CA), and ZIPB differs from Zip14 withrespect to nearly every property tested, including its pH de-pendence, temperature dependence, saturation kinetics, sub-strate profile, and anion dependence (24), providing no basisfor anticipating that ZIPB and Zip14 share a common mecha-nism. Moreover, currents observed for ZIPB were specific forzinc and were obtained with millimolar zinc concentrations andin the absence of most physiological ions (24).

Two murine models of the acute-phase response have dem-onstrated the inducibility of Zip14 in the liver (27). IL-6,IL-1�, and nitric oxide are among the mediators of Zip14

expression in hepatocytes and lead to Zip14-dependent zincaccumulation (23, 27). ChIP assays have shown AP-1 associ-ation with the Zip14 promoter and Zip14 hnRNA, indicative oftranscription, is increased by nitric oxide (23). Under suchconditions there is an increase in Zip14 associated with theplasma membrane. These responses have been interpreted asphysiological homeostatic responses to stresses and infections.How such responses are factored into pathophysiological out-comes, such as iron-overload disorders, warrants investigation.

pH dependence of Zip14-mediated Fe2� transport and itsimplications for cell-specific iron and zinc transport. Our dataindicated that Zip14 transports iron within a narrow pH range(pH � 6.5) and optimally at pH � 7.5. We therefore expectthat wherever Zip14 is expressed on plasma membranes, suchas is observed in hepatocytes (25), this transporter should becapable of mediating cellular uptake of NTBI characteristic ofiron-overload conditions. The observation that zinc potentlyinhibits iron uptake suggests that Zip14 expressed at theplasma membrane should primarily serve zinc transport undernormal conditions.

Zip14 may also participate in transferrin-associated ironuptake in hepatocytes (47) by mobilizing iron from earlyendosomes to cytoplasm. Suppression of Zip14 expression bysiRNA resulted in the inhibition of iron assimilation in HepG2cells by �50% (47). Iron is liberated from the transferrin-transferrin receptor complex after only relatively modest en-dosomal acidification (50% dissociation at pH � 6.5; Ref. 36),and we expect Zip14 to be functional, albeit suboptimally, atpH 6.5. In contrast, DMT1, which is H� coupled and maxi-mally stimulated at low pH (Fig. 5) (30, 31), may serve as thepredominant route of iron mobilization from late endosomesand lysosomes, and DMT1 is essential for erythroid ironassimilation (17).

Our previous study indicated that, in mice, Zip14 is mosthighly expressed in the small intestine among the tissues tested(25). Although its plasma-membrane localization and apical/basolateral distribution in intestine is yet to be established,Zip14 could play a role in zinc and iron acquisition in the

Fig. 10. Zip14-dependent Ca2� uptake and the effects of divalent metal ions.A: uptake of 150 �M 45Ca2� in the absence (None) or presence of 30 �M Fe2�

or 30 �M Cd2� in control oocytes (gray bars) and oocytes expressing Zip14(black bars, n � 9–12) in media containing 1 mM L-ascorbic acid and 100 �Mniflumic acid. Two-way ANOVA revealed an interaction (P 0.001); withinZip14, anot significant (unadjusted P � 0.68) and bP 0.001 cf. “None”.B: uptake of 300 �M 45Ca2� in the absence (None) or presence of 15 �M Zn2�

in control oocytes (gray bars) and oocytes expressing Zip14 (black bars, n �12–13) in media containing 100 �M niflumic acid. Two-way ANOVA re-vealed an interaction (P 0.001).

Fig. 9. Metal-ion inhibition profiles of Zip14-mediated Cd and Mn transport.A: uptake of 2 �M 109Cd2� in the absence (None) or presence of a range ofcandidate inhibitor metal ions each at 20 �M, in the presence of 1 mML-ascorbic acid, in control oocytes (gray bars), and oocytes expressing Zip14(black bars) (n � 8–15). Within Zip14, Cd2� and Zn2� inhibited 109Cd2�

uptake (unadjusted P 0.001); all other comparisons cf. “None” were notsignificant (unadjusted P � 0.031). B: uptake of 2 �M 54Mn2� in the absence(None) or presence of a range of candidate inhibitor metal ions each at 20 �M,in the presence of 1 mM L-ascorbic acid, in control oocytes (gray bars), andoocytes expressing Zip14 (black bars) (n � 10–15). Within Zip14, each metalion inhibited 54Mn2� uptake (unadjusted P 0.001).



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neonate before the onset of expression of intestinal Na�/H�

exchangers (4) around weaning. Thereafter, the maturation ofthe acidic microclimate (�pH 6.0) at the mammalian intestinalbrush border (32, 33) should be expected to suppress Zip14iron-transport activity (see Figs. 4C and 5). In contrast, DMT1,the principal mechanism by which nonheme iron is taken up atthe intestinal brush border (reviewed in Refs. 21 and 29), isrequired to maintain iron homeostasis in the adult (17) but isnot essential for iron acquisition in the suckling mammal (43).Since Zip14-mediated Zn2� transport was active over abroader pH range (see Fig. 8D) than was Fe2� transport, Zip14may also contribute to intestinal Zn2� transport in the adult;however, its activity is not sufficient to compensate for loss ofZIP4 in acrodermatitis enteropathica (10). This zinc malab-sorption disorder is corrected with oral zinc therapy, demon-strating the ability of other transporters to supply essentialamounts of zinc during diminished ZIP4 transport activity, andzinc absorption appears to be served by a multiplicity oftransporters (7, 22).

In conclusion, our study reveals that Zip14 is a complexmetal-ion transporter whose broad substrate profile includesCd2�, Fe2�, Mn2�, and Zn2�. Whereas Zip14 may serve totransport Fe2� out of transferrin-containing endosomes inhepatocytes (47), we speculate that Zn2� would be the pre-dominant physiological substrate of Zip14 wherever it is ex-pressed at the plasma membrane. Zip14 efficiently transportedCd2�, so Zip14 should be considered a candidate mechanismof cellular uptake in cadmium exposure. Our observation ofincomplete mutual inhibition for Zip14 raises the possibilitythat additional iron transporters may be hiding in the ZIP(SLC39) family of metal-ion transporters.

ACKNOWLEDGMENTS

We thank Sarah R. Anthony, Jesse M. Ewald, and Yossief Haileab (Uni-versity of Cincinnati) for help in the laboratory, and Drs. Elizabeta Nemeth andBo Qiao (David Geffen-UCLA School of Medicine) for graciously providingthe EGFP-tagged DMT1.

This work was presented in part at Experimental Biology, April 18–22,2009 at New Orleans, LA (39).

Present address of N. Zhao: Dept. of Cell and Developmental Biology,Oregon Health & Science Univ., Portland, OR 97239. Present address of J. P.Liuzzi: Dept. of Dietetics and Nutrition, Robert Stempel School of PublicHealth and Social Work, Florida International Univ., Miami, FL 33199.

GRANTS

This study was supported by Grants R01 DK-080047 (to B. Mackenzie),R01 DK-080706 (to M. D. Knutson), R01 DK031127 (to R. J. Cousins), andP30 DK-078392 (Digestive Health Center, Cincinnati Children’s Hospital andUniversity of Cincinnati) from the National Institute of Diabetes and Digestiveand Kidney Diseases (NIDDK) and by the University of Cincinnati (to B.Mackenzie). The production of copper-64 at Washington University-St. LouisSchool of Medicine is supported by Grant R24 CA086307 from the NationalCancer Institute (NCI). The content of this study is solely the responsibility ofthe authors and does not necessarily represent the official views of the NCI,NIDDK, or the National Institutes of Health.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

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Zip14 is a complex broad-scope metal-ion transporter whose