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This Article Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Illing, A. C Articles by Mackenzie, B. PubMed Articles by Illing, A. C Articles by Mackenzie, B.

Substrate profile and metal-ion selectivity of the human divalent metal-ion transporter DMT1

Dept Mol & Cell Physiology, University of Cincinnati, PO Box 670576, Cincinnati, OH, 45267-0576

ABSTRACT

DMT1 is essential for intestinal iron absorption and erythroid iron utilization. Whereas DMT1 exhibits reactivity (based on evoked currents) with a broad range of transition metal ions, questions have arisen as to which of these are actually transported. We used a fluorescence-based approach to image metal-ion transport in Xenopus oocytes and provide here the first comprehensive substrate profile analysis for human DMT1. We established the reactivity of the fluorophore PhenGreen SK (PGSK) with a range of metal ions in a cell-free system. We injected oocytes with PGSK and monitored fluorescence changes during 10-min superfusion with metal ions. We took the first-order rate constant of quenching as an index of metal-ion uptake at pH 5.5 and validated our approach by comparing our findings with radiotracer data. The K_0.5 for Fe²⁺ (4.2 ± 2.2 µM) determined from PGSK quenching matched that determined from ⁵⁵Fe²⁺ uptake (5.9 ± 0.9 µM). Neither Cr²⁺ nor Cr³⁺ quenched PGSK fluorescence, consistent with ⁵¹Cr data revealing that Cr²⁺ and Cr³⁺ are excluded by DMT1. DMT1 also excluded Cu¹⁺, Cu²⁺, Fe³⁺, Sn²⁺, Sr²⁺ and V²⁺. Several metal ions resulted in more rapid quenching in DMT1 oocytes than in controls. We determined for these the order of substrate selectivity (using the ratio I_max/K_0.5) from evoked currents: Fe²⁺, Cd²⁺ > Co²⁺, Mn²⁺ > Ni²⁺, Pb²⁺, V³⁺, Zn²⁺ (> 0.5 log₁₀ units). ⁵⁵Fe²⁺ transport was competitively inhibited by Cd²⁺, Mn²⁺ and Co²⁺. Whereas Zn²⁺ evoked large currents, it was poorly transported and only weakly inhibited ⁵⁵Fe²⁺ transport (K_i 50 µM). Our data reveal that DMT1 is an iron-preferring transporter that is a likely route of entry for the toxic heavy metal Cd. DMT1 may also contribute to the absorption of Co and Mn (and trace metals Ni and V) but is unlikely to be physiologically relevant to the absorption of Zn or Cu.

This Article Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Illing, A. C Articles by Mackenzie, B. PubMed Articles by Illing, A. C Articles by Mackenzie, B.