Belgrade rats exhibit microcytic, hypochromic anemia and systemic iron 
deficiency due to a glycine-to-arginine mutation at residue 185 in a metal ion 
transporter of a divalent metal transporter/divalent cation transporter/solute 
carrier 11 group A member 2 or 3 (DMT1/DCT1/SLC11A2), a member of the 
natural-resistance-associated macrophage protein (Nramp) family. By use of 
rabbit duodenal tissue, a calcein fluorescence assay has previously been 
developed to assess transport of divalent metal ions across the small-intestinal 
brush border membrane (BBM). The assay was readily applied here to rat BBM to 
learn if it detects DMT1 activity. The results demonstrate protein-mediated 
transport across the BBM of all tested ions: Mn(2+), Fe(2+), and Ni(2+). 
Transport into BBM vesicles (BBMV) from (b/b) Belgrade rats was below the 
detection limit. BBMV of +/b origin had substantial activity. The kinetic rate 
constant for Ni(2+) membrane transport for +/b BBMV was within the range for 
normal rabbit tissue. Vesicles from +/b basolateral membranes (BLM) showed 
similar activity to BBMV while b/b BLM vesicles (BLMV) lacked transport 
activity. Immunoblots using isoform-specific antibodies demonstrated that 
intestinal levels of b/b DMT1 were increased compared to +/b DMT1, reflecting 
iron deficiency. Immunoblots on BBMV indicated that lack of activity in b/b 
vesicles was not due to a failure of DMT1 to localize to the BBMV; an excess of 
specific isoforms was present compared to +/b BBMV or duodenal extracts. 
Immunoblots from BLMV also exhibited enrichment in DMT1 isoforms, despite their 
distinct origin. Immunofluorescent staining of thin sections of b/b and +/b 
proximal intestines confirmed that DMT1 localized similarly in mutant and 
control enterocytes and showed that DMT1 isoforms have distinct distributions 
within intestinal tissue.