IRON AND ZINC TRANSPORT : OXIDATIVE STRESS-MODULATION OF DCT1

In humans, iron plays a key role in a number of metabolic functions. Its deficiency as well as its overload leads to important pathologies. Excess of free iron can amplify the effect of oxidative stress, via Fenton's reaction, associated with a great number of diseases such as ischemia-reperfusion injury, inflammation, carcinogenesis and degenerative diseases of the SNC. The functional properties and the localisation of the divalent cation transporter (DCT1/Nramp2/DMT1) in recycling endosomes and in plasma membranes indicate its key role in the direct cellular iron uptake and in the transferrin-receptor mediates pathway. Though DCT1 expression is regulated at a post-transcriptional level via the IRP/IRE system, in order to prevent iron overload in oxidative stress condition, a direct regulation of the protein in the plasma membrane could represent an important defensive mechanism of the cell. To study the influence of redox reagents on DCT1 function we used the Xenopus laevis oocyte expression system and analysed the transport activity with radiotracer 55Fe2+ and 65Zn2+ -uptake assays and electrophysiology experiments. Our radiotracer experiments confirmed the capability of DCT1 to mediate Zn uptake previously suggested on the basis of electrophysiology experiments. Since the characteristics of Zn2+ induced DCT1 mediated current resembled the characteristics of the iron current, we performed our experiments with Zn2+ which does not react with the used redox reagents. Hydrogen peroxide treatment resulted in an inhibition of about 40 % of both Zn2+ uptake and Zn2+ induced DCT1 mediated current in oocytes. DCT1 sequence contains several amino acid residues that could be targets for oxidant reagents. Therefore Hg2+ administration exerted the same inhibition than H2O2 and, in both cases, DCT1 function was fully recovered after perfusion with dithiotreitol strongly indicating the involvement of cysteine residues. Mutagenesis experiments allowed us to identify a cysteine that is involved in the mechanism of DCT1 inhibition. C248 is located in the forth-outer loop of the predicted secondary structure of the transporter and could represent a sensor that allows the cell to activate defensive mechanism. In fact reactive oxygen species can be released from some kind of cells like phagocytes to affect target cells. The finding of a direct inhibition of iron transport appears to have relevant physiological interest to understand the regulation of iron metabolism and the defensive mechanisms to preserve cell from oxidative injury.