Human ferroportin (FPN) transports proton along the iron gradient. (A) Assay of proton transport based on the quenching of ACMA fluorescence. ACMA is provided from the outside to diffuse into the liposome²²  (black arrow). After proton is bound, ACMA cannot diffuse out. To promote proton transport by FPN, 10 μM Fe²⁺ is provided at the outside to generate an influx gradient. The positive charge (blue sphere) introduced by Fe²⁺ and H⁺ influx is compensated for by K⁺ outflow through valinomycin (blue arrow). (B) Proton transport by FPN requires a Fe²⁺ gradient. Addition of Fe²⁺ at 5 minutes and CCCP at 25 minutes are indicated above the curves, and valinomycin (Val) is added at 0 minutes. (C) Proton transport by FPN in the presence of a Co²⁺ gradient. To generate an influx gradient, 100 μM Co²⁺ is provided from the outside of the liposome. Other experimental setups are the same as in panel B.