OXIDATIVE STRESS MODULATION OF DCT1

Iron plays a key role in a number of metabolic functions. Its deficiency as well as its overload leads to important pathologies in humans. The functional properties and the localization of the divalent cation transporter (DCT1/Nramp2/DMT1) indicate its weight in the direct cellular iron uptake and in the transferrin-receptor mediated pathway (H.Gunshin et al. Nature 388: 482-488, 1997). To study the influence of redox reagents on DCT1 function we used the Xenopus l. oocyte expression system and analyzed the transport activity with iron and zinc uptake assays, and electrophysiology experiments. Our radiotracer experiments confirmed the capability of DCT1 to mediate Zn uptake suggested on the basis of electrophysiology experiments. Therefore we used Zn2+, which does not react with redox reagents used in our study. Hydrogen peroxide treatment resulted in an inhibition of about 40% of zinc induced DCT1 mediated current in oocytes. DCT1 sequence contains several amino acid residues that could be targets for oxidant reagents. 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 involved in the mechanism of DCT1 inhibition. C248 is thought to be located in the third-outer loop of the predicted secondary structure of the transporter and could represent a sensor that allows the cell to prevent iron overload in oxidative-stress conditions. 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