Human ferroportin (FPN) supports uphill active transport. (A) Assay of proton transport with an uphill Fe²⁺ gradient. The gradient is generated by enclosing 100 μM Fe²⁺ inside the liposome and providing 1 μM Fe²⁺ from the outside. The influx of Fe²⁺ and H⁺ is promoted by a charge gradient that is generated from the K⁺ outflow through valinomycin (Val; blue arrow). The assay of proton transport by the quenching of ACMA fluorescence is the same in Figure 4. (B) FPN transports protons against an uphill Fe²⁺ gradient. Addition of Fe²⁺ and CCCP are indicated above the curves. (C) FPN transports protons against an uphill Co²⁺ gradient, with 100 μM Co²⁺ enclosed inside the liposome and 1 μM Co²⁺ provided from the outside.