Molecular impact of human divalent metal‐ion transporter DMT1 mutations associated with disease phenotypes

DMT1 is a H + ‐coupled divalent metal‐ion transporter critical for intestinal iron absorption and erythroid iron utilization. Recent reports describe two unrelated cases in which mutations in human DMT1 were associated with severe microcytic anemia and hepatic iron overload. We are using voltage‐clamp and radiotracer assays in Xenopus oocytes expressing mutant or wildtype (wt) DMT1 to examine the molecular impact of such mutations and explore DMT1 structure‐function. E399D‐DMT1 functions like wtDMT1 except for a modest reduction in apparent affinity for Fe 2+ ( K 0.5 = 2.1 ± 0.3 μM; wtDMT1, 1.0 ± 0.4 μM), whereas the phenotype of the patient homozygous for this mutation likely arises from the increased frequency of an aberrant splice variant lacking exon 12. Compound heterozygous mutations in a second patient comprise a 3‐bp deletion in intron 4 and a substitution (R416C) at a conserved amino acid residue. R416C significantly lowers Fe 2+ transport activity in oocytes without a loss of affinity for Fe 2+ or H + , and does not alter substrate profile. The reduced V max results both from reduced plasma membrane expression and reduced turnover rate of the transport cycle (14 s −1 ) compared with wtDMT1 (23 s −1 , 30 °C), however, 30‐35% of the mRNA in this patient also arises from abnormal splicing that deletes 40 amino acids. Whereas the functional consequences of coding‐sequence point mutations E399D and R416C at best only partially explain the probands’ phenotypes, such mutations may offer new clues in the structure‐function of DMT1.