Hepcidin Inhibits Ferroportin Activity Via a Rapid, Low‐Affinity, and Calcium‐Dependent Mechanism in Xenopus O ocytes Independent of Endocytosis

Abstract

The cellular iron-exporter ferroportin (Fpn), under the control of the hormone hepcidin, serves as the key control point in iron homeostasis. The canonical mechanism by which hepcidin suppresses Fpn activity comprises hepcidin binding by Fpn and the subsequent internalization and degradation of the transporter. Here we explored hepcidin inhibition of 55Fe efflux from RNA-injected Xenopus oocytes expressing human Fpn. Hepcidin pretreatment rapidly (t½ = 10 ± [SEM] 0.3 min; r2 = 0.99, P < 0.001) inhibited Fpn-mediated 55Fe efflux at low affinity (EC50 = 0.5 ± 0.06 µM; r2 = 0.91, P < 0.001). In contrast, hepcidin-induced Fpn internalization in mammalian cells exhibits EC50 in the low nanomolar range. Hepcidin did not induce Fpn internalization, a conclusion based on the following observations: (i) Hepcidin action was only modestly temperature-dependent (temperature quotient, Q10 = 3.14 ± 0.25 over the range 16–26°C) whereas endocytosis is expected to be strongly temperature-dependent, i.e. Q10 in the order of 5–17 [Weigel PH and Oka JA (1981) J Biol Chem 256, 2615–17] (ii) Inhibition of Fpn by hepcidin proceeded even in the presence of blockers of clathrin-mediated (chlorpromazine) and clathrin-independent (filipin, genistein) endocytosis, and (iii) Hepcidin treatment produced no meaningful change in either the abundance of protein at the plasma membrane or membrane surface area (as determined by capacitance) in oocytes expressing Fpn, despite reducing Fpn-mediated iron transport by 65%. Hepcidin had no effect on Fpn activity when we removed calcium from the pretreatment medium (0 Ca2+, 1 mM EGTA). Our data reveal that hepcidin can directly block Fpn activity via a rapid, low-affinity, Ca2+-dependent mechanism that does not involve endocytosis. We speculate that the Xenopus oocyte lacks the specific signal by which hepcidin triggers Fpn internalization. The Xenopus oocyte is therefore an ideal model system in which to study the mechanism by which hepcidin directly blocks Fpn activity.