Iron and copper transport activities of the mammalian metal‐ion transporters DMT1 and CTR1

Divalent metal‐ion transporter‐1 (DMT1) and the high‐affinity copper transporter‐1 (CTR1) play essential roles in the intestinal absorption of iron and copper respectively. Both transporters are potential therapeutic targets in disorders of metal metabolism. Disagreement remains over whether the metal‐ion specificities of the two transporters overlap. For example, others have concluded that DMT1 transports Cu + and CTR1 transports Fe 2+ based on the results of shRNA knockdown of gene expression in Caco‐2 cells [Espinoza A et al (2012) Biol Trace Elem Res 146, 281]. Since these observations contradicted our published data regarding the substrate profile of DMT1 in vitro [Illing AC et al (2012) J Biol Chem 287, 30485] and in vivo [Shawki A et al (2015) Am J Physiol Gastrointest Physiol 309, G635] or of CTR1 in vitro [Lee J et al (2002) J Biol Chem 277, 4380], we have re‐examined metal‐ion transporter substrate profiles. We tested the hypothesis that DMT1 can transport copper and that CTR1 can transport iron by using radiotracer assays and the voltage clamp in RNA‐injected oocytes expressing human DMT1 or CTR1. Expression of CTR1 in oocytes stimulated up to 20‐fold the uptake of 2 μM 64 Cu + (pH 5.5) but did not stimulate the uptake of 64 Cu 2+ or 55 Fe 2+ . Expression of DMT1 in oocytes stimulated 80‐fold the uptake of 2 μM 55 Fe 2+ (pH 6.0) but transport of 64 Cu + or 64 Cu 2+ did not differ from that in control oocytes. We found that Cu + inhibited DMT1‐mediated 55 Fe 2+ uptake in a competitive fashion ( K i ≈ 58 μM). In DMT1‐expressing oocytes, Cu + evoked inward currents; however, such currents were also observed in control oocytes and their I / V relationships did not resemble those for DMT1 substrates Fe 2+ and Cd 2+ . Addition of Fe 2+ or Cd 2+ abolished the presteady‐state (transient) currents observed in the absence of metal ion whereas addition of Cu + or Cu 2+ did not. We found, however, that Cu 2+ accelerated the decay of the transient currents. We conclude that (1) whereas Cu + and Cu 2+ are capable of interacting with DMT1, neither are transported substrates; and (2) CTR1 mediates transport of Cu + but not of Cu 2+ or Fe 2+ . We therefore find no evidence of overlapping metal‐ion specificities in DMT1 and CTR1. Support or Funding Information PHS grants R01‐DK080047, R01‐DK107309, R01‐DK074192, and R25‐HL115473; and the American Physiological Society