Comparison of mammalian cell lines expressing distinct isoforms of divalent metal transporter 1 in a tetracycline-regulated fashion

Abstract

DMT1 (divalent metal transporter; also known as SLC11A2, DCT1 or Nramp2) is responsible for ferrous iron uptake in the duodenum, iron exit from endosomes during the transferrin cycle and some transferrin-independent iron uptake in many cells. Four protein isoforms differ by starting in exon 1A or 2 and ending with alternative peptides encoded by mRNA that contains or lacks an IRE (iron responsive element; +/-IRE). We have compared 1A/+IRE and 2/-IRE DMT1 during regulated ectopic expression. HEK-293-F (human embryonic kidney-293-fast growing variant) cells were stably transfected with each construct expressed from a tetracycline-regulated CMV promoter. Reverse transcriptase-PCR analysis showed that construct expression responded to doxycycline. Immunofluorescence staining of cells, using antibodies specific for DMT1 isoforms, confirmed an increase in expression in the plasma membrane and cytosolic vesicles after doxycycline treatment, but with isoform specific distributions. Immunoblotting also revealed stimulation of expression. Nevertheless, both DMT1 isoforms performed similarly in assays for functional properties based on 54Mn2+ and 59Fe2+ uptake. Mn incorporation after doxycycline treatment was approximately 10-fold greater than that of untreated cells, while expression in the untreated cells was approximately 5-fold greater than in the untransfected cells. Uptake of Mn depended on addition of doxycycline, with half maximal response at approximately 1 nM doxycycline. Doxycycline-stimulated Mn and Fe uptake was linear with time for 10 min but not over longer periods. Transport exhibited a pH optimum at approximately 5.5 and dependence on incubation temperature and Mn or Fe concentration. The new cell lines should prove useful for research on metal homoeostasis, toxicological studies and efforts to identify distinctive properties of the isoforms.

Divalent metal transporter, DMT1: A novel MRI reporter protein

“…All cell lines showed some response to increasing levels of Mn, but naive HEK cells and the knockdown cell line were relatively insensitive compared to the +DMT1 cells, which demonstrated up to a 4-fold increase in R 1 above control HEK cells in 300μM Mn. This substantial shift in R 1 was consistent with a large increase in Mn uptake caused by DMT1 overexpression, as shown in previous studies (16).…”

“…In the nervous system, MEMRI has been developed for both activity imaging, based on Mn uptake through Ca channels, and tract tracing based on the axonal transport of Mn (12,13,14,15). However, Ca channels are not the sole means of Mn uptake in cells; indeed, DMT1 has been shown to actively transport Mn and other divalent metals (16), and the high affinity of DMT1 for Mn has been characterized in several tissues (17). Based on this observation, we hypothesized that engineering the expression of DMT1 would result in detectable contrast in vivo.…”

Human iron transporters

“…Potentially, differences between the N-terminus that starts in exon 1A and the one that starts in exon 2 and differences between the 18 C-terminal amino acid residues of the ?IRE form and the 25 of the -IRE could affect localization of the transporter within the cell, thus where it functions and even where it turns over. Existing data do suggest the presence of such distinctions [5,15,24,26,38,39,48,49].…”

Calcium-channel blockers do not affect iron transport mediated by divalent metal-ion transporter-1

“…Since their paper disagreed with our published data 4,5 regarding the activities of the DMT1 isoforms (described previously [4][5][6] ), we undertook to resolve that issue and examine the effect of nifedipine on DMT1 in vitro.…”