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

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 55Fe in the presence of l-ascorbate but not nitrilotriacetic acid, indicating that Zip14 is an iron transporter specific for ferrous ion (Fe2+) over ferric ion (Fe3+). Zip14-mediated 55Fe2+ uptake was saturable ( K0.5 ≈ 2 μM), temperature-dependent (apparent activation energy, Ea = 15 kcal/mol), pH-sensitive, Ca2+-dependent, and inhibited by Co2+, Mn2+, and Zn2+. HCO3− stimulated 55Fe2+ 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 109Cd2+, 54Mn2+, and 65Zn2+ but not 64Cu (I or II). 65Zn2+ uptake also was saturable ( K0.5 ≈ 2 μM) but, notably, the metal-ion inhibition profile and Ca2+ dependence of Zn2+ transport differed from those of 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, we found that Zip14 is capable of mediating cellular uptake of NTBI characteristic of iron-overload conditions.

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Functional properties of multiple isoforms of human divalent metal-ion transporter 1 (DMT1)

Biochemical Journal ◽

10.1042/bj20061290 ◽

2007 ◽

Vol 403 (1) ◽

pp. 59-69 ◽

Cited By ~ 119

Author(s):

Bryan Mackenzie ◽

Hitomi Takanaga ◽

Nadia Hubert ◽

Andreas Rolfs ◽

Matthias A. Hediger

Keyword(s):

Plasma Membrane ◽

Functional Properties ◽

Metal Ion ◽

Cell Types ◽

Divalent Metal ◽

The Body ◽

Ion Transporter ◽

Voltage Dependent ◽

Divalent Metal Ion ◽

Responsive Element

DMT1 (divalent metal-ion transporter 1) is a widely expressed metal-ion transporter that is vital for intestinal iron absorption and iron utilization by most cell types throughout the body, including erythroid precursors. Mutations in DMT1 cause severe microcytic anaemia in animal models. Four DMT1 isoforms that differ in their N- and C-termini arise from mRNA transcripts that vary both at their 5′-ends (starting in exon 1A or exon 1B) and at their 3′-ends giving rise to mRNAs containing (+) or lacking (−) the 3′-IRE (iron-responsive element) and resulting in altered C-terminal coding sequences. To determine whether these variations result in functional differences between isoforms, we explored the functional properties of each isoform using the voltage clamp and radiotracer assays in cRNA-injected Xenopus oocytes. 1A/IRE(+)-DMT1 mediated Fe2+-evoked currents that were saturable (K0.5Fe≈1–2 μM), temperature-dependent (Q10≈2), H+-dependent (K0.5H≈1 μM) and voltage-dependent. 1A/IRE(+)-DMT1 exhibited the provisional substrate profile (ranked on currents) Cd2+, Co2+, Fe2+, Mn2+>Ni2+, V3+≫Pb2+. Zn2+ also evoked large currents; however, the zinc-evoked current was accounted for by H+ and Cl− conductances and was not associated with significant Zn2+ transport. 1B/IRE(+)-DMT1 exhibited the same substrate profile, Fe2+ affinity and dependence on the H+ electrochemical gradient. Each isoform mediated 55Fe2+ uptake and Fe2+-evoked currents at low extracellular pH. Whereas iron transport activity varied markedly between the four isoforms, the activity for each correlated with the density of anti-DMT1 immunostaining in the plasma membrane, and the turnover rate of the Fe2+ transport cycle did not differ between isoforms. Therefore all four isoforms of human DMT1 function as metal-ion transporters of equivalent efficiency. Our results reveal that the N- and C-terminal sequence variations among the DMT1 isoforms do not alter DMT1 functional properties. We therefore propose that these variations serve as tissue-specific signals or cues to direct DMT1 to the appropriate subcellular compartments (e.g. in erythroid cells) or the plasma membrane (e.g. in intestine).

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