Although manganese is an essential trace metal, little is known about its transport and homeostatic regulation. Here we have identified a cohort of patients with a novel autosomal recessive manganese transporter defect caused by mutations in SLC39A14. Excessive accumulation of manganese in these patients results in rapidly progressive childhood-onset parkinsonism–dystonia with distinctive brain magnetic resonance imaging appearances and neurodegenerative features on post-mortem examination. We show that mutations in SLC39A14 impair manganese transport in vitro and lead to manganese dyshomeostasis and altered locomotor activity in zebrafish with CRISPR-induced slc39a14 null mutations. Chelation with disodium calcium edetate lowers blood manganese levels in patients and can lead to striking clinical improvement. Our results demonstrate that SLC39A14 functions as a pivotal manganese transporter in vertebrates.

Here, we directly tested the hypothesis that Zip14 transports free zinc, iron, and other metal ions by using the Xenopus laevis oocyte heterologous expression system, and use of this approach also allowed us to characterize the functional properties of Zip14. Expression of mouse Zip14 in RNAinjected oocytes stimulated the uptake of 55 Fe in the presence of L-ascorbate but not nitrilotriacetic acid, indicating that Zip14 is an iron transporter specific for ferrous ion (Fe 2ϩ ) over ferric ion (Fe 3ϩ uptake also was saturable (K0.5 Ϸ 2 M) but, notably, the metal-ion inhibition profile and Ca 2ϩ dependence of Zn 2ϩ transport differed from those of Fe 2ϩ transport, and we propose a model to account for these observations. Our data reveal that Zip14 is a complex, broad-scope metal-ion transporter. Whereas zinc appears to be a preferred substrate under normal conditions, we found that Zip14 is capable of mediating cellular uptake of NTBI characteristic of iron-overload conditions. cadmium transport; hereditary hemochromatosis; iron; homeostasis iron transport; Xenopus laevis oocyte; SLC39A14; thalassemia; zinc transport IRON-OVERLOAD CONDITIONS (e.g., thalassemia, hereditary hemochromatosis) are characterized by the appearance in plasma of nontransferrin-bound iron (NTBI) and result in cardiomyopathy, diabetes, hepatic cancer, and cirrhosis. Identification of the routes of cellular NTBI uptake will therefore provide novel targets for therapeutics.Zrt-and Irt-like protein-14 (Zip14) is a member of a large family of mammalian metal-ion transporters, the SLC39 gene family (6,11,12,22,26). Zip14 (synonyms SLC39A14, KIAA0062) is strongly expressed in the intestine (25, 35) but its subcellular localization there is not yet clear. Notably, Zip14 is abundantly expressed in the liver, heart, and pancreas (25, 35, 42), the major sites of organ damage in iron overload. Our previous data identified Zip14 as a candidate route for NTBI uptake since overexpression of Zip14 in human embryonic kidney (HEK)293, SF9, or HeLa cell lines stimulated NTBI uptake (14, 25), whereas small interfering RNA (siRNA) suppression of endogenous Zip14 in AML12 mouse hepatocytes decreased NTBI uptake (25).We have expressed mouse Zip14 in RNA-injected Xenopus oocytes, an efficient heterologous expression system ideal for direct assays of membrane transport and tolerant of broad manipulation of experimental conditions. We used radiotracer assays to test the hypothesis that Zip14 transports free iron and to examine the functional properties and metal-ion substrate profile of Zip14. MATERIALS AND METHODSReagents. Restriction enzymes were obtained from New England Biolabs (Ipswich, MA). All other reagents were obtained from SigmaAldrich (St. Louis, MO) or Research Products International (Prospect, IL) unless otherwise indicated.Expression of mouse Zip14 and human DMT1 in Xenopus oocytes. We performed laparotomy and ovariectomy on adult female Xenopus laevis frogs (Nasco, Fort Atkinson, WI) under 3-aminoethylbenzoate methanesulfonate anesthesia...

Discovery and function of hepcidin in iron homeostasisHepcidin is a key peptide hormone that regulates iron homeostasis in chordates. Hepcidin was initially characterized as an antimicrobial peptide in a mass spectrometry-based search for cysteine-rich defensin-like peptides in blood (1) and in urine (2). However, both groups showed that hepcidin displays only a weak antimicrobial activity. Further, unlike defensins, which vary in sequence among species, hepcidin is highly conserved from zebrafish to humans. Shortly after hepcidin was described, subtractive hybridization studies comparing the livers of normal and iron overloaded mice established a connection between iron loading and increased hepcidin mRNA (3, 4). The fundamental insight into hepcidin's role in iron homeostasis came in 2004 with the discovery that hepcidin acts to lower iron in the blood by binding to and downregulating the iron transporter, ferroportin (FPN1) (5). FPN1 is the only known transporter that is responsible for the efflux of iron from cells. Downregulation of FPN1 by hepcidin in splenic and hepatic macrophages decreases the ability of macrophages to export recycled iron from senescent rbcs, which constitute the primary source of iron in the plasma. In addition, a high concentration of hepcidin in the blood decreases the transport of iron out of intestinal epithelial cells, further limiting iron in the blood. Control of hepcidin expressionHepcidin is synthesized, processed to an active form, and secreted predominantly by hepatocytes (6, 7). The expression of hepcidin is mediated through the bone morphogenetic protein (BMP) and JAK2/STAT3 signaling pathways (Figure 1). Under nonpathological conditions, iron levels in the body upregulate hepcidin expression. Although the underlying mechanisms are poorly understood, recent studies have documented the essential roles of hemojuvelin (HJV), hereditary hemochromatosis protein (HFE), transferrin receptor 2 (TfR2), and matriptase-2 (MT2, encoded by the gene TMPRSS6) in the process of hepcidin regulation in humans and animal models as well as of BMP6, neogenin, and BMP receptors (ActRIIA/ALK2/ALK3) in animal models (8)(9)(10)(11)(12)(13)(14)(15)(16)(17).Intact BMP signaling is essential for hepcidin expression. The canonical BMP-signaling pathway is initiated upon BMP binding to a BMP receptor complex on the cell surface, which activates the receptor kinase to phosphorylate the cytoplasmic proteins SMAD1, SMAD5, and SMAD8. These phosphorylated, receptor-regulated SMADs then form transcription factor complexes with SMAD4, consisting of receptor-regulated SMADs and SMAD4, that translocate into the nucleus to induce the transcription of target genes such as hepcidin (18). In mice, liver-specific disruption of SMAD4 or the BMP receptors ALK2 or ALK3 markedly decreased hepcidin expression, resulting in iron overload (15,19).BMP6. At least 20 BMPs are expressed in mammals. In vitro studies reveal that BMP2, -4, -5, -6, -7, and -9 can robustly induce BMP signaling and markedly increase hepcidin expres...

ZIP14 is a transmembrane metal ion transporter that is abundantly expressed in the liver, heart, and pancreas. Previous studies of HEK 293 cells and the hepatocyte cell lines AML12 and HepG2 established that ZIP14 mediates the uptake of nontransferrin-bound iron, a form of iron that appears in the plasma during pathologic iron overload. In this study we investigated the role of ZIP14 in the cellular assimilation of iron from transferrin, the circulating plasma protein that normally delivers iron to cells by receptor-mediated endocytosis. We also determined the subcellular localization of ZIP14 in HepG2 cells. We found that overexpression of ZIP14 in HEK 293T cells increased the assimilation of iron from transferrin without increasing levels of transferrin receptor 1 or the uptake of transferrin. To allow for highly specific and sensitive detection of endogenous ZIP14 in HepG2 cells, we used a targeted knock-in approach to generate a cell line expressing a FLAG-tagged ZIP14 allele. Confocal microscopic analysis of these cells detected ZIP14 at the plasma membrane and in endosomes containing internalized transferrin. HepG2 cells in which endogenous ZIP14 was suppressed by siRNA assimilated 50% less iron from transferrin compared with controls. The uptake of transferrin, however, was unaffected. We also found that ZIP14 can mediate the transport of iron at pH 6.5, the pH at which iron dissociates from transferrin within the endosome. These results suggest that endosomal ZIP14 participates in the cellular assimilation of iron from transferrin, thus identifying a potentially new role for ZIP14 in iron metabolism. Most cells acquire iron from transferrin (TF),2 a circulating plasma protein that can carry up to two ferric (Fe 3ϩ ) iron atoms. After Fe-TF binds to cell surface TF receptor 1 (TFR1), the plasma membrane invaginates into clathrin-coated pits, which internalize the Fe-TF⅐TFR1 complex into endosomes. Upon endosomal acidification, Fe 3ϩ is released and subsequently reduced to Fe 2ϩ . The liberated Fe 2ϩ is then transported across the endosomal membrane and into the cytosol (1).The assimilation of iron from TF has been best characterized in developing erythroid cells, the most avid consumers of TFbound iron (TBI). In these cells, reduction of Fe 3ϩ is catalyzed by the oxidoreductase Steap3 (2), and iron transport out of the endosome is facilitated by the transmembrane protein divalent metal transporter 1 (DMT1) (3, 4). Accordingly, mice lacking either Steap3 or DMT1 cannot incorporate sufficient iron into developing erythrocytes and become anemic (2, 3). After the erythroid marrow, the second largest consumer of TBI is the liver, accounting for 10 -20% of iron exchange with the plasma (5). Interestingly, anemic Steap3-mutant mice or DMT1-null mice are able to take up iron into the liver (6, 7), indicating that Steap3 and DMT1 are dispensable for hepatic iron uptake.Under normal conditions, Ͼ95% of plasma iron is TBI. Studies in perfused rat liver document that the liver takes up TBI, almost exclusively into he...

ZIP14 (encoded by the solute carrier 39 family member 14 [SLC39A14] gene) is a manganese (Mn) transporter that is abundantly expressed in the liver and small intestine. Loss-of-function mutations in SLC39A14 cause severe hypermanganesemia. As the liver is regarded as the main regulatory organ involved in Mn homeostasis, impaired hepatic Mn uptake for subsequent biliary excretion has been proposed as the underlying disease mechanism. However, liver-specific Zip14 knock-out (KO)