期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 275, 期 46, 页码 35738-35745出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M005387200
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资金
- NIAID NIH HHS [1 RO1 AI35237-08] Funding Source: Medline
Nrampa, also known as DMT1 and DCT1, is a la-transmembrane (TM) domain protein responsible for dietary iron uptake in the duodenum and iron acquisition from transferrin:in peripheral tissues. Nramp2/DMT1 produces by alternative splicing two isoforms differing at their C terminus (isoforms I and II). The subcellular localization, mechanism of action, and destination of divalent cations transported by the two Nrampa isoforms are not completely understood. Stable CHO transfectants expressing Nramp2 isoform II modified by addition of a hemaglutinin epitope in the loop defined by the TM7-TM8 interval were generated. Immunofluorescence with permeabilized and intact cells established that Nrampa isoform II is expressed at the plasma membrane and demonstrated the predicted extracytoplasmic location of the TM7-TR18 loop. Using the fluorescent, metal-sensitive dye calcein, and a combination of membrane-permeant and -impermeant iron chelators, Nrampa transport was measured and quantitated with respect to kinetic parameters and at steady state. Iron 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-accessible pool. Parallel experiments with transfectants expressing the lysosomal Nramp1 homolog do not show any divalent cation transport activity, establishing major functional differences between Nramp1 and Nramp2. Monitoring the effect of Nramp2 on the calcein-sensisitve labile iron pool allows a simple, rapid, and nonisotopic approach to the functional study of this protein.
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