Transport of divalent transition-metal ions is lost in small-intestinal tissue of b/b Belgrade rats.
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.
Knöpfel
Martin
M
Department of Biochemistry, SUNY, Buffalo, New York 14214-3000, USA.
Zhao
Lin
L
Garrick
Michael D
MD
D000818:Animals; D001692:Biological Transport / Q000235:genetics; D027682:Cation Transport Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002413:Cations, Divalent / Q000378:metabolism; D005260:Female; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D007413:Intestinal Mucosa / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D007421:Intestine, Small / Q000737:chemistry / Q000378:metabolism*; D007501:Iron / Q000378:metabolism; D033862:Iron-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008297:Male; D008345:Manganese / Q000378:metabolism; D019216:Metals, Heavy / Q000378:metabolism*; D018613:Microscopy, Confocal; D008871:Microvilli / Q000378:metabolism; D009532:Nickel / Q000378:metabolism; D020033:Protein Isoforms / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344; D011922:Rats, Mutant Strains; D017208:Rats, Wistar; D022161:Transport Vesicles / Q000378:metabolism; D000099236:Solute Carrier Family 11, Member 2
D027682:Cation Transport Proteins; D002413:Cations, Divalent; D007501:Iron; D033862:Iron-Binding Proteins; D008345:Manganese; D019216:Metals, Heavy; D009532:Nickel; D020033:Protein Isoforms; D000099236:Solute Carrier Family 11, Member 2