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J. Biol. Chem., Vol. 275, Issue 46, 35738-35745, November 17, 2000
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From the Department of Biochemistry, McGill University, Montreal,
Quebec H3G 1Y6, Canada
Nramp2, also known as DMT1 and DCT1, is a
12-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 Nramp2 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
Nramp2 isoform II is expressed at the plasma membrane and demonstrated
the predicted extracytoplasmic location of the TM7-TM8 loop. Using the
fluorescent, metal-sensitive dye calcein, and a combination of
membrane-permeant and -impermeant iron chelators, Nramp2 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
Nramp2 expression. Iron uptake by Nramp2 at the plasma membrane was
into the nonferritin-bound, calcein-accessible so-called "labile iron
pool." Ion selectivity experiments show that Nramp2 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.
Nramp 2 (DCT1/DMT1) Expressed at the Plasma Membrane Transports
Iron and Other Divalent Cations into a Calcein-accessible
Cytoplasmic Pool*
*
This work was supported by National Institutes of Health
Grant 1 RO1 AI35237-08 (to P. G.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
International Research Scholar of the Howard Hughes Medical
Institute. Senior Scientist of the Medical Research Council of Canada.
To whom correspondence should be addressed: Dept. of Biochemistry, McGill University, 3655 Drummond, Rm. 907, Montreal, PQ H3G 1Y6, Canada. Tel.: 514-398-7291; Fax: 514-398-2603; E-mail:
gros@med.mcgill.ca.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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