Molecular impact of a human divalent metal‐ion transporter‐1 (DMT1) mutation (G212V) found in two compound heterozygotes with microcytic anemia (893.40)

DMT1 is a H + ‐coupled divalent metal‐ion transporter that is critical for intestinal iron (Fe) absorption and erythroid Fe utilization. Beaumont et al ( Blood 107 , 4168 [2006]) and Bardou‐Jacquet et al ( Blood Cells Mol Dis 47 , 243 [2011]) have described two patients compound heterozygous for mutations in DMT1 associated with hypochromic–microcytic anemia and hepatic Fe overload. The unrelated probands have in common a G212V substitution; the first proband also possesses a V114 deletion and the second, an N491S substitution. The probands’ siblings and parents each possess one mutated allele but were reportedly 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 mutations. The V114 deletion and N491S substitution abolished 55 Fe 2+ transport activity whereas 55 Fe 2+ transport mediated by G212V‐DMT1 did not differ from wtDMT1. G212V had no effect on the pH dependence of 55 Fe 2+ transport, presteady‐state kinetics, or apparent affinity for Fe 2+ ( K 0.5 = 1.2 ± [SE] 0.4 µM cf. 1.5 ± 0.2 µM in wtDMT1; P = 0.55, n = 6 oocytes); however, G212V mediated much smaller Fe 2+ ‐evoked currents than did wtDMT1. From simultaneous measurement of currents and 55 Fe 2+ fluxes in wtDMT1, we determined the H + /Fe 2+ coupling ratio to be 17 ± 1 which greatly exceeds that expected for a tightly coupled system, i.e., most of the wtDMT1‐mediated current arises from H + slippage. In contrast, the H + /Fe 2+ coupling ratio for G212V was 1 ± 0.3. Slippage in cotransporters is normally thought of in terms of the energetic penalty; however, our observation that H + slippage, but not Fe 2+ transport, is disrupted in a mutant associated with disease raises the possibility that H + slippage serve a physiological role. Grant Funding Source : Supported by PHS Grant DK080047