Molecular impact of divalent metal‐ion transporter (DMT1) mutations (V114del and G212V) found in a compound heterozygote with microcytic anemia and hepatic iron overload

Abstract

DMT1 is a H+‐coupled divalent metal‐ion transporter critical for intestinal iron (Fe) absorption and erythroid Fe utilization. Beaumont et al (Blood 107, 4168 [2006]) described a patient compound heterozygous for two new DMT1 mutations (V114 deletion and G212V) associated with hypochromic‐microcytic anemia and hepatic Fe overload. The proband's sibling and parents each possess one mutated allele but were asymptomatic. We expressed mutant and wildtype (wt) DMT1 in Xenopus oocytes and used voltage‐clamp and radiotracer assays to examine the molecular impact of these novel mutations. The V114 deletion abolished 55Fe2+ transport activity whereas 55Fe2+ transport mediated by G212V‐DMT1 did not differ from wtDMT1. G212V had no effect on the pH dependence of 55Fe2+ transport, presteady‐state kinetics, or the affinity for Fe2+ (K0.5 = 1.7 ± 1.1 μM c.f. 1.3 ± 0.5 μM in wtDMT1). To our surprise, however, G212V‐DMT1 mediated much smaller Fe2+‐evoked currents than did wtDMT1. From simultaneous measurement of currents and 55Fe2+ fluxes, we arrived at a wildtype H+/Fe2+ ratio of 17 ± 1 which greatly exceeds that expected for strict stoichiometric transport, i.e. most of the wtDMT1‐mediated current arises from H+ slippage. Such slippage is normally thought of in terms of the energetic penalty to the cell; however, indications that H+ slippage, but not Fe2+ transport, is disrupted in the G212V mutant associated with disease raise the possibility that H+ slippage serve a physiological role.