Nramp 2 (DCT1/DMT1) expressed at the plasma membrane transports iron and other divalent cations into a calcein-accessible cytoplasmic pool

Nrampa, also known as DMT1 and DCT1, is a la-transmembrane (TM) domain prot ein responsible for dietary iron uptake in the duodenum and iron acquisitio n from transferrin:in peripheral tissues. Nramp2/DMT1 produces by alternati ve splicing two isoforms differing at their C terminus (isoforms I and II). The subcellular localization, mechanism of action, and destination of diva lent cations transported by the two Nrampa isoforms are not completely unde rstood. Stable CHO transfectants expressing Nramp2 isoform II modified by a ddition of a hemaglutinin epitope in the loop defined by the TM7-TM8 interv al were generated. Immunofluorescence with permeabilized and intact cells e stablished that Nrampa isoform II is expressed at the plasma membrane and d emonstrated the predicted extracytoplasmic location of the TM7-TR18 loop. U sing the fluorescent, metal-sensitive dye calcein, and a combination of mem brane-permeant and -impermeant iron chelators, Nrampa transport was measure d and quantitated with respect to kinetic parameters and at steady state. I ron transport at the plasma membrane was time- and pH-dependent, saturable, and proportional to the amount of Nrampa expression. Iron uptake by Nrampa at the plasma membrane was into the nonferritin-bound, calcein-accessible so-called "labile iron pool." Ion selectivity experiments show that Nrampa isoform II can also transport Co2+ and Cd2+ but not Mg2+ into the calcein-a ccessible pool. Parallel experiments with transfectants expressing the lyso somal Nramp1 homolog do not show any divalent cation transport activity, es tablishing major functional differences between Nramp1 and Nramp2. Monitori ng the effect of Nramp2 on the calcein-sensisitve labile iron pool allows a simple, rapid, and nonisotopic approach to the functional study of this pr otein.