Zip14 is a complex broad-scope metal-ion transporter whose functional properties support roles in the cellular uptake of zinc and nontransferrin-bound iron

Abstract

Recent studies have shown that overexpression 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 heterologous expression system, and use of this approach also allowed us to characterize the functional properties of Zip14. Expression of mouse Zip14 in RNA-injected oocytes stimulated the uptake of (55)Fe in the presence of l-ascorbate but not nitrilotriacetic acid, indicating that Zip14 is an iron transporter specific for ferrous ion (Fe(2+)) over ferric ion (Fe(3+)). Zip14-mediated (55)Fe(2+) uptake was saturable (K(0.5) ≈ 2 μM), temperature-dependent (apparent activation energy, E(a) = 15 kcal/mol), pH-sensitive, Ca(2+)-dependent, and inhibited by Co(2+), Mn(2+), and Zn(2+). HCO(3)(-) stimulated (55)Fe(2+) transport. These properties are in close agreement with those of NTBI uptake in the perfused rat liver and in isolated hepatocytes reported in the literature. Zip14 also mediated the uptake of (109)Cd(2+), (54)Mn(2+), and (65)Zn(2+) but not (64)Cu (I or II). (65)Zn(2+) uptake also was saturable (K(0.5) ≈ 2 μM) but, notably, the metal-ion inhibition profile and Ca(2+) dependence of Zn(2+) transport differed from those of Fe(2+) 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, we found that Zip14 is capable of mediating cellular uptake of NTBI characteristic of iron-overload conditions.

Fig. 1.

Imaging of enhanced green fluorescent protein (EGFP)-Zrt, Irt-like protein 14 (Zip14), and divalent metal-ion transporter 1 (DMT1)-EGFP expression in Xenopus oocytes. Confocal laser-scanning microscopy of control oocytes and oocytes expressing EGFP-Zip14 or DMT1-EGFP. Representative images are presented in which the optical slice (7.3 μm at ×10 magnification or 0.6 μm at ×40) approximately bisects the oocyte. Scale bars (white) indicate 0.2 mm.

Fig. 2.

Western blot analysis of membrane fractions from Xenopus oocytes expressing EGFP-Zip14 and DMT1-EGFP by using anti-green fluorescent protein (GFP) antibody. Each lane (numbered at top) was loaded with membrane fractions (∼3 μg protein per lane) isolated from the following: lane 1, control oocytes; lanes 2 and 3, oocytes expressing DMT1-EGFP; lanes 4 and 5, DMT1; lanes 6 and 7, EGFP-Zip14; lanes 8 and 9, Zip14. Intensities of the immunoreactive bands in each lane of the Western blot were normalized by quantity of protein loaded in each lane determined by densitometric analysis of reversible Ponceau staining (40) of the blot (not shown).

Fig. 3.

Zip14 mediates cellular uptake of free iron. A: time course of uptake of 2 μM ⁵⁵Fe²⁺ (in the presence of 1 mM l-ascorbic acid) in control oocytes and oocytes 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 and y-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/min and y-intercept 0.02 ± 0.01 pmol (adjusted r² = 0.99; P < 0.001). B: ⁵⁵Fe²⁺ saturation kinetics in the range 0.2–50 μM Fe²⁺ in the presence of 1 mM l-ascorbic acid (n = 14–16). Data for Zip14 (black symbols and lines) were fit by Eq. 1 yielding parameters V_max^Fe = 1.1 ± 0.1 pmol/min, n_H^Fe = 1.1 ± 0.2, K_0.5^Fe = 2.3 ± 0.5 μM (adjusted r² = 0.97, P < 0.001). ⁵⁵Fe²⁺ uptake was measured at 0.2 and 50 μM Fe²⁺ in control oocytes (gray symbols and lines) and joined by a linear fit. C: uptake of 2 μM ⁵⁵Fe from media containing 1 mM nitrilotriacetic acid (NTA) (in place of l-ascorbic acid, l-Asc) or 1 mM l-ascorbic acid in control oocytes and oocytes expressing Zip14 (n = 11–13). Two-way ANOVA revealed an interaction (P < 0.001); within NTA, Zip14 did not differ from control (unadjusted P = 0.90).

Fig. 4.

Properties of Zip14-mediated Fe²⁺ transport. A: metal-ion inhibition profile of Zip14-mediated ⁵⁵Fe²⁺ transport. Uptake of 2 μM ⁵⁵Fe²⁺ in the absence (None) or presence of a range of candidate inhibitor metal ions each at 20 μM, in the presence of 1 mM l-ascorbic acid, in control oocytes (gray bars) and oocytes expressing Zip14 (black bars) (n = 10–14). Within Zip14, all metals inhibited ⁵⁵Fe²⁺ uptake (P < 0.001). B: temperature (T) dependence of Zip14-mediated uptake of 2 μM ⁵⁵Fe²⁺ (n = 9–13). Data were fit by Eq. 2 to obtain activation energy (E_a) = 15.2 ± 2.0 kcal/mol, ln(A) = 25.0 ± 3.5 (adjusted r² = 0.90, P < 0.001). For clarity, control data are not displayed. C: uptake of 2 μM ⁵⁵Fe²⁺ as a function of extracellular pH in oocytes expressing Zip14 (black symbols and line, n = 27–30). Within Zip14, uptakes at each pH differed from one another (unadjusted P < 0.007) except pH 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 5.5 and 8.5 (n = 31–32). Zip14 did not differ from control at pH 5.5 (unadjusted P = 0.77) but did at pH 8.5 (unadjusted P = 0.004).

Fig. 5.

Comparison of the iron-transport activities of mouse Zip14 and human DMT1. Uptake of 2 μM ⁵⁵Fe²⁺ was measured at pH 5.5 and 7.5 in control oocytes (gray bars) and oocytes expressing mouse Zip14 (black bars) or human DMT1 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. control at pH 7.5; DMT1 at pH 5.5 differed from Zip14 at pH 7.5 (unadjusted P < 0.001).

Fig. 6.

Ion dependence of Zip14-mediated ⁵⁵Fe²⁺ transport. A: effect of bicarbonate (HCO₃⁻) on Zip14-mediated uptake of 2 μM ⁵⁵Fe²⁺ (n = 10–11) measured over 2 min to minimize pH changes with time; two-way ANOVA revealed an interaction (P < 0.001). B: effect of Cl⁻ replacement with isethionate (Iseth) on Zip14-mediated uptake of 2 μM ⁵⁵Fe²⁺ (n = 12–15); two-way ANOVA revealed no effect of Cl⁻ replacement (P = 0.58) and no interaction (P = 0.58). C: uptake of 2 μM 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 V_max^Fe = 1.0 ± 0.1 pmol/min, K_d^Ca = 0.39 ± 0.12 mM (adjusted r² = 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 metal ions (*Me²⁺, each at 2 μM in the presence of 1 mM l-ascorbic acid) in control oocytes and oocytes expressing Zip14 (n = 17–27). Two-way ANOVA revealed 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 μM ⁶⁴Cu was measured in the presence of 1 mM l-histidine and in the presence (⁶⁴Cu¹⁺) or absence (⁶⁴Cu²⁺) of 1 mM l-ascorbic acid and compared with uptake of 2 μM ⁵⁵Fe²⁺ in the presence of 1 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 not differ from control for ⁶⁴Cu¹⁺ (P = 1.0) or ⁶⁴Cu²⁺ (P = 0.56) but differed for ⁵⁵Fe²⁺ (P < 0.001).

Fig. 8.

Properties of ⁶⁵Zn²⁺ transport. A: ⁶⁵Zn²⁺ saturation kinetics in the range 0.1–10 μM Zn²⁺ (n = 10–11). Data for Zip14 (black symbols and line) were fit by Eq. 1 yielding parameters V_max^Zn = 1.8 ± 0.2 pmol/min, n_H^Zn = 0.9 ± 0.1, K_0.5^Zn = 1.9 ± 0.6 μM (adjusted r² = 0.99, P < 0.001). ⁶⁵Zn²⁺ uptake was measured at 0.1, 1.0, and 10 μM 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 μM ⁶⁵Zn²⁺ in the absence (None) or presence of a range of candidate inhibitor metal ions each at 20 μM, in the presence of 1 mM l-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 of Zip14-mediated uptake of 2 μM ⁶⁵Zn²⁺ (n = 10–11). Data were fit by Eq. 2 to obtain E_a = 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 μM ⁶⁵Zn²⁺ as a function of extracellular pH in oocytes expressing Zip14 (black symbols and 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, unadjusted P > 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 symbols and 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 (unadjusted P < 0.001). E: uptake of 2 μM 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).

Fig. 9.

Metal-ion inhibition profiles of Zip14-mediated Cd and Mn transport. A: uptake of 2 μM ¹⁰⁹Cd²⁺ in the absence (None) or presence of a range of candidate inhibitor metal ions each at 20 μM, in the presence of 1 mM l-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 not significant (unadjusted P > 0.031). B: uptake of 2 μM ⁵⁴Mn²⁺ in the absence (None) or presence of a range of candidate inhibitor metal ions each at 20 μM, in the presence of 1 mM l-ascorbic acid, in control oocytes (gray bars), and oocytes expressing Zip14 (black bars) (n = 10–15). Within Zip14, each metal ion inhibited ⁵⁴Mn²⁺ uptake (unadjusted P < 0.001).

Fig. 10.

Zip14-dependent Ca²⁺ uptake and the effects of divalent metal ions. A: uptake of 150 μM ⁴⁵Ca²⁺ in the absence (None) or presence of 30 μM Fe²⁺ or 30 μM 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 μM niflumic acid. Two-way ANOVA revealed an interaction (P < 0.001); within Zip14, ^anot significant (unadjusted P = 0.68) and ^bP < 0.001 cf. “None”. B: uptake of 300 μM ⁴⁵Ca²⁺ in the absence (None) or presence of 15 μM Zn²⁺ in control oocytes (gray bars) and oocytes expressing Zip14 (black bars, n = 12–13) in media containing 100 μM niflumic acid. Two-way ANOVA revealed an interaction (P < 0.001).