Mechanism of Ca2+ transport by ferroportin

Introduction

Ferroportin (Fpn), encoded by the SLC40A1 gene, is the only known Fe²⁺ exporter in human (Nemeth and Ganz, 2021; Abboud and Haile, 2000; Donovan et al., 2000; McKie et al., 2000). Fpn is highly expressed on enterocytes, hepatocytes, and macrophages, and mediates release of iron stored in cells (Drakesmith et al., 2015; Mackenzie and Garrick, 2005; Knutson et al., 2005). Activity of Fpn is regulated by hepcidin, a small peptide hormone secreted by hepatocytes, which binds to Fpn and reduces iron export by inhibiting its transport activity and triggering endocytosis of Fpn (Aschemeyer et al., 2018; Nemeth et al., 2004; Nemeth and Ganz, 2021). Mutations in Fpn cause hereditary hemochromatosis and iron-loading anemias in human (Pietrangelo, 2017; Ganz, 2013; Ginzburg, 2019; Vlasveld et al., 2019).

We have shown previously that Fpn is a Fe²⁺/2H⁺ exchanger, that is, the export of one Fe²⁺ is accompanied by the import of two H⁺ (Pan et al., 2020). The electroneutral transport mechanism is likely an adaptation to overcome the negative resting membrane potential that would have significantly hindered export of cations. Structures of mammalian Fpn show two transition-metal ion binding sites, termed Site 1 (S1) and Site 2 (S2) (Billesbølle et al., 2020; Pan et al., 2020). Curiously, each ion binding site is composed of only two residues, Asp39 and His43 for S1, and Cys326 and His507 for S2, and it remains unsettled how S1 and S2 mediate coupled transport of Fe²⁺ and H⁺. In the current study, we found that Asp39 is also part of a Ca²⁺ binding site, and that mutation Asp39Ala almost completely eliminates Ca²⁺ transport but has a modest impact on Fe²⁺ transport, suggesting that S1 and S2 may have different roles in Fe²⁺ or H⁺ transport.

Ca²⁺ is known to bind to mammalian Fpn (Deshpande et al., 2018), but Ca²⁺ transport by Fpn has not been demonstrated and the role of Ca²⁺ in Fe²⁺ transport remains ambiguous. Using Fpn expressed in Xenopus oocytes, Deshpande et al. found that Fe²⁺ export requires Ca²⁺, but Ca²⁺ is not transported by Fpn (Deshpande et al., 2018). More recently, several studies using purified Fpn...