Non-transferrin-bound iron transporters

Author’s Accepted Manuscript Non-transferrin-bound iron transporters Mitchell D. Knutson

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S0891-5849(18)32176-2 https://doi.org/10.1016/j.freeradbiomed.2018.10.413 FRB13977

To appear in: Free Radical Biology and Medicine Received date: 15 August 2018 Revised date: 4 October 2018 Accepted date: 8 October 2018 Cite this article as: Mitchell D. Knutson, Non-transferrin-bound iron transporters, Free Radical Biology and Medicine, https://doi.org/10.1016/j.freeradbiomed.2018.10.413 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Non-transferrin-bound iron transporters Mitchell D. Knutson Food Science and Human Nutrition Department, University of Florida, Gainesville, FL, USA Email: [email protected] ABSTRACT Most cells in the body acquire iron via receptor-mediated endocytosis of transferrin, the circulating iron transport protein. When cellular iron levels are sufficient, the uptake of transferrin decreases to limit further iron assimilation and prevent excessive iron accumulation. In iron overload conditions, such as hereditary hemochromatosis and thalassemia major, unregulated iron entry into the plasma overwhelms the carrying capacity of transferrin, resulting in non-transferrin-bound iron (NTBI), a redox-active, potentially toxic form of iron. Plasma NTBI is rapidly cleared from the circulation primarily by the liver and other organs (e.g., pancreas, heart, and pituitary) where it contributes significantly to tissue iron overload and related pathology. While NTBI is usually not detectable in the plasma of healthy individuals, it does appear to be a normal constituent of brain interstitial fluid and therefore likely serves as an important source of iron for most cell types in the CNS. A growing body of literature indicates that NTBI uptake is mediated by non-transferrin-bound iron transporters such as ZIP14, L-type and T-type calcium channels, DMT1, ZIP8, and TRPC6. This review provides an overview of NTBI uptake by various tissues and cells and summarizes the evidence for and against the roles of individual transporters in this process. Graphical abstract

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Abbreviations CNS, central nervous system; CSF, cerebrospinal fluid; DMT1, divalent metal-ion transporter-1; IRE, iron-responsive element; LPI, labile plasma iron; LTCC L-type calcium channel; NMDA, N-methyl-Daspartate; NTBI, non-transferrin-bound iron; SLC39A8, solute carrier family 39 member 8; SLC39A14, solute carrier family 39 member 14; TTCC, T-type calcium channel; TRPC6, transient receptor potential cation channel subfamily C member 6; VGCCs, voltage-gated calcium channels; ZIP8, ZRT/IRT-like protein 8; ZIP14, ZRT/IRT-like protein 14;

Keywords Hereditary hemochromatosis; thalassemia major; labile plasma iron; ZIP14, L-type calcium channels

1. Introduction Iron in blood plasma is tightly bound to transferrin, the circulating transport protein that delivers iron throughout the body. Cells take up transferrin-bound iron in proportion to the number of cell-surface transferrin receptors. The expression of transferrin receptors, in turn, is regulated by cellular iron status, which allows cells to take up iron in proportion to their needs. Greater than 80% of plasma transferrinbound iron is taken up by developing red blood cells of the bone marrow, where iron is utilized in the synthesis of heme for hemoglobin. Usually, only about one-third of all transferrin molecules are carrying iron, which gives a large buffering capacity in case plasma iron concentrations increase. In iron overload disorders such as hereditary hemochromatosis or thalassemia major, the amount of iron in plasma often exceeds the carrying capacity of transferrin, giving rise to what is known as “non-transferrin-bound iron” or “NTBI”. Plasma NTBI is efficiently cleared from the circulation by the liver, pancreas, heart, and various other endocrine organs, where it contributes significantly to tissue iron accumulation, thus increasing the risk of iron-related pathology [1]. At the cellular level, NTBI uptake is mediated by one or more cell membrane transport proteins that can be collectively referred to as NTBI transporters, although they are known to serve more primary physiological functions in the transport of iron or other metals. This review summarizes the evidence for and against the roles of these transporters in NTBI uptake, with special emphasis on tissues and cells most affected by iron overload. Relevant background on NTBI and quantitative aspects of its uptake by various tissues is also provided. 2. Plasma NTBI: definition and measurement NTBI was first identified in 1978 by Hershko et al. [1], who described a fraction of low-molecularweight, chelatable iron that is not bound to transferrin in the plasma of patients with iron overload. This non-specific pool of plasma iron was considered non-physiological because it could not be detected in the plasma of healthy individuals. More recent definitions describe NTBI as “circulating iron that is not bound to transferrin, ferritin, or heme” [3]. Although NTBI is generally implicated in tissue iron accumulation and toxicity, some of this NTBI may be bound non-specifically to plasma proteins and may therefore not be redox active or available for transport into cells [2]. These ambiguities have given rise to the term “labile plasma iron” (LPI), which refers to the fraction of plasma NTBI that is both redox active and chelatable—and therefore most pertinent pathologically and pharmacologically [3].

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Remarkably, 40 years after its identification, the exact chemical composition of NTBI remains poorly characterized. This is probably because NTBI represents a heterogeneous and variable mix of Fe3+ bound to small molecules such as citrate, acetate, and phosphate [4]. Of these potential iron-binding ligands, citrate is the most abundant and has the highest affinity for Fe3+ [5]. Indeed, the presence of Fe3+-citrate has been demonstrated by NMR and HPLC analysis of plasma from hemochromatosis patients with iron overload and fully saturated transferrin [6]. A higher molecular weight fraction of NTBI has also been described, which likely includes Fe2+or Fe3+ complexes bound to albumin [4]. It has been postulated that the different forms of NTBI vary with the degree and type of iron overload disease [7]. Given the heterogeneous nature of NTBI in plasma, its measurement is fraught with technical difficulties, and no standardized method exists. A recent international round robin of 10 different leading assays and plasma samples representing 10 different disease states reported plasma NTBI concentrations as high as 15 mol/L and plasma LPI concentrations as high as 2.5 mol/L [8]. Although absolute values of NTBI/LPI varied among assays, NTBI and LPI were readily detected when transferrin saturations exceeded 70% and 90%, respectively, with values increasing hyperbolically as transferrin saturation increased [8]. As a point of reference, levels of plasma iron (i.e., iron bound to transferrin) typically range from 10–30 mol/L. In iron overload, plasma transferrin concentrations usually decrease, but the amount of iron bound to transferrin can still reach levels as high as ~45 mol/L [8]. 3. Measurement of NTBI uptake in vivo Tissue uptake of NTBI in laboratory animals is usually measured after intravenous administration [9-14] or orogastric gavage [11, 12, 15] of radiolabeled iron (e.g., 59Fe-labeled ferric citrate). Intravenous (systemic) administration provides the highest bioavailability because it avoids the first-pass effect of hepatic metabolism. Orogastric administration allows one to determine the disposition of iron ingested orally, but can be complicated by variability in enteral absorptive processes. In either case, plasma transferrin must be saturated to ensure that the administered 59Fe does not bind to apo-transferrin. In normal animals, plasma transferrin can be transiently saturated by intravenous injection of nonradioactive iron 10 minutes before 59Fe administration. Using this technique in mice, Craven et al. [11] determined that the plasma clearance of NTBI is 30 seconds—much faster than that of transferrin-bound iron (50 minutes). Hypotransferrinemic mice, which have < 1% of normal circulating levels of transferrin [16], have also been used to assess tissue uptake of NTBI [9-11, 17] 4. Tissue uptake of plasma NTBI: the main organs The vast majority of plasma NTBI is taken up by the liver, the principal site of iron storage in the body. Pioneering research in the 1960s demonstrated that when plasma transferrin of rats or dogs is saturated with iron, >80% of 59Fe given orally or directly into the portal vein is deposited in the liver [15]. Subsequent studies using perfused rat liver showed that 60–75% of NTBI is removed in a single pass, with most of the iron depositing in hepatocytes [18, 19]. The major role of the liver in clearing orally derived NTBI has been additionally documented in humans [20, 21], hypotransferrinemic mice [11], and hemojuvelin knockout mice [12]. Indeed, the highly efficient hepatic first-pass extraction of NTBI from portal blood explains why the liver is the primary organ that loads iron in hereditary hemochromatosis. In this disease, plasma transferrin is fully saturated and therefore any iron entering the portal circulation, from either the diet or erythrocyte-derived iron recycled by the spleen, will reach the liver as NTBI. When NTBI is administered intravenously, the liver is still the main organ of NTBI clearance, but uptake by the pancreas and kidney becomes more evident. For example, two hours after tail vein administration of 59Fe-NTBI to mice, approximately 60% of the injected 59Fe dose was recovered in the liver, while 7–

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10% was found in the kidneys, and 5–7% in the pancreas [12, 14]. In iron overload, the pancreas loads iron predominantly in acinar cells and to a lesser degree in beta cells [22-24]. Interestingly, mice show no or very little beta cell iron accumulation even under extreme conditions of iron overload [25-28]. In the kidney, 59Fe-NTBI administered to mice is initially detected in the cortex and then deposits in the medulla [29], consistent with the model that plasma NTBI, either as ferric citrate or iron bound to albumin, is filtered by the glomerulus and deposits in the distal nephron [30]. Indeed, microinjection of 55FeCl3 reveals that, within the kidney tubule, most NTBI is taken up by the distal nephron [31]. Accordingly, mouse models of iron overload (hepcidin or hemojuvelin knockout mice) accumulate iron predominantly in epithelial cells of the distal tubule, most notably in the thick ascending limb [30]. Although the heart is well known to take up NTBI, it does so considerably less well, taking up only about 1% of 59Fe-NTBI intravenously administered to mice [12]. When expressed per gram of tissue, the heart takes up approximately one-third as much 59Fe-NTBI as does the pancreas [10]. In iron overload, iron accumulates in cardiomyocytes, which appear to be particularly susceptible to iron toxicity [32]. Comparisons of NTBI and TBI uptake in cultured rat cardiomyocytes demonstrates that NTBI uptake is 100-300 times greater than uptake of iron from transferrin [33, 34], and that NTBI uptake is stimulated by iron loading, suggesting positive-feedback regulation [35, 36]. The relative efficiencies of NTBI uptake by the liver, pancreas, and heart are consistent with the time course of tissue iron accumulation: iron first loads in the liver followed by the pancreas and heart [37, 38]. Analysis of tissue iron loading from 3 to 52 weeks of age in three mouse models of hereditary hemochromatosis suggests that iron starts to load significantly in the pancreas and heart after the liver has reached its iron-storage capacity [37, 38]. Moreover, iron contents of the pancreas and heart at 52 weeks of age correlated strongly in all three mouse models, suggesting a common source of iron loading. Correlations between pancreatic and cardiac iron have also been demonstrated in thalassemia major patients, with significant cardiac iron deposition occurring nearly a decade later than pancreatic iron [38]. The time course of iron accumulation in the kidney has been less well documented. However, in one study of hypotransferrinemic mice, kidney iron concentrations increased in parallel with those of the heart and pancreas [39]. 5. NTBI uptake by endocrine tissues In iron overload disorders in which iron loads rapidly and massively (e.g., juvenile hemochromatosis and thalassemia major), endocrine complications are often the first clinical manifestations of the disease [4042]. These complications include hypogonadotropic hypogonadism, reduced glucose tolerance/diabetes, adrenal insufficiency, and hypothyroidism, which are associated with iron accumulation in the anterior pituitary gland, pancreatic beta cells, adrenal gland, and thyroid gland, respectively. Although NTBI uptake by these tissues has not been directly measured in vivo, it is generally believed that NTBI is a main contributor to endocrine iron accumulation as it is elsewhere [1]. NTBI uptake has been documented in isolated human islets and lox5 cells, a human beta cell line [43]. Of all the endocrine complications associated with iron overload, hypogonadotropic hypogonadism is by far the most common, affecting 70–80% of thalassemia major patients worldwide [44] and 77% of patients with juvenile hemochromatosis in one study [41]. Hypogonadotropic hypogonadism results from insufficient pituitary production of luteinizing hormone and follicle-stimulating hormone and is characterized by delayed or absent sexual development. Although the etiology of hypogonadotropic hypogonadism is likely multifactorial, iron toxicity to gonadotroph cells of the anterior pituitary is thought to be primarily responsible as the pituitary can load pathological amounts of iron early in life [44, 45]. Using magnetic resonance imaging to measure pituitary iron, Noetzli et al. [46] found that 25% of

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thalassemia major patients displayed severe pituitary iron deposition by the age of 10. Analyses of the anterior pituitary from patients with iron overload reveals that iron loads in all five cell types (gonadotrophs, thyrotrophs, corticotrophs, somatotrophs, lactotrophs), with heaviest iron deposits in gonadotrophs [45]. Pathological iron accumulation in pituitary thyrotrophs, which secrete thyroid stimulating hormone, has been postulated to contribute to secondary (central) hypothyroidism in patients with thalassemia major [47], although primary hypothyroidism caused by abnormalities of the thyroid gland is more common. Iron-related thyroid complications have been attributed to iron deposition in the follicular epithelium [45]. In the adrenal gland, iron has been reported to load mostly in the zona glomerulosa, the site of mineralocorticoid synthesis [48]. 6. NTBI uptake into and within the brain Compared to most other organs, the brain is relatively resistant to iron overload. This is generally thought to be because the brain is protected by two barriers—the blood-brain barrier and the blood-CSF barrier. Nonetheless, modest brain iron accumulation has been documented in iron-loaded mice [25, 49] and in patients with thalassemia major [50, 51]. Studies in Hpx mice unequivocally demonstrate that NTBI can be taken up into the brain [9, 17, 52]. In wild-type mice injected with 59Fe-NTBI, the 59Fe is readily detectable in the ventricles within 2 hours, and after 24 hours, it is present diffusely throughout the brain parenchyma. In the ventricles, the 59Fe concentrates in the choroid plexus, suggesting that this structure is involved in iron delivery to the brain [53]. Iron accumulation in the choroid plexus has been observed in mouse models of iron overload [25]. Within the brain, NTBI is considered to be a physiologic form of iron because transferrin is fully saturated in the CSF (and likely also in the interstitial fluid), even under normal conditions [54]. Indeed, NTBI appears to be the main, if not exclusive, source of iron for astrocytes, oligodendrocytes, and microglia, as these cell types do not express transferrin receptor in vivo [55]. NTBI uptake has been studied extensively in primary cultures of astrocytes [56-63], which have been proposed to play a protective role in the brain by scavenging NTBI [64]. NTBI uptake has also been amply studied in neurons [65-71], yet less so in microglia [72, 73]. Bishop et al. [72] compared NTBI uptake among various neural cell types and found that microglia were most efficient at accumulating NTBI, followed by astrocytes, and neurons. 7. Non-Transferrin-Bound Iron Transporters Proteins identified as NTBI transporters are shown in Table 1. The evidence supporting a role for each transporter in NTBI uptake differs considerably, with some transporters having support only from cell culture studies (e.g., ZIP8 and TRPC6) while others have direct in vivo support from knockout mouse models (e.g., ZIP14). Studies using inhibitors (e.g., calcium channel blockers) provide abundant in vivo support for a role for LTCCs/TTCCs, whereas inhibitors to ZIP14 and ZIP8 have yet to be developed. TRPC6 was first implicated in NTBI transport in 2004, but few supporting studies have been published since then. 7.1 ZIP14 (SLC39A14) ZIP14 belongs to the ZRT/IRT-like protein (ZIP) family, which consists of 14 members in mammals [74]. ZIP14 is predicted to have 8 transmembrane domains, a long extracellular N-terminal region, and a short extracellular C-terminus. Transmembrane domains IV and V are notably amphipathic and have metalbinding histidine and glutamic acid residues that have been proposed to form part of a transport channel

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[75]. Among human tissues, ZIP14 is ubiquitously expressed, with most abundant expression in liver, pancreas, and heart [76, 77]. Similar tissue expression profiles for ZIP14 are found in mouse, although one study reported highest ZIP14 expression in the duodenum [78]. ZIP14 was first identified as a cellsurface zinc transporter [76], but subsequent studies showed that it also transports iron [78]. The idea that ZIP14 transports iron is not surprising given that the founding member of the ZIP family is IRT1 (IronRegulated Transporter 1), an Arabidopsis protein that plays an essential role in iron uptake from the soil [79]. A link between ZIP14 and NTBI uptake was hypothesized [78] based on the observation that ZIP14 in human tissues is most abundantly expressed in the liver, pancreas, and heart—organs that notably accumulate iron and show iron-related pathology. Consistent with this hypothesis, siRNA-mediated suppression of ZIP14 expression in a mouse hepatocyte cell line diminished the cellular uptake of 59Fe from ferric citrate [78]. Functional studies of ZIP14 expressed in Xenopus oocytes revealed that ZIP14mediated Fe2+ transport was saturable, temperature dependent, Ca2+ dependent, optimal at pH 7.5, and inhibited by Co2+, Mn2+, and Zn2+ [80]. All of these transport characteristics agree with previous studies of NTBI uptake in perfused rat liver [19]. Moreover, the observation that ZIP14 levels are elevated in iron-loaded rat liver and pancreas demonstrates that ZIP14 is regulated by iron [81]. Iron-loaded tissue samples from infants with neonatal hemochromatosis have been reported to stain strongly for ZIP14, with colocalization between ZIP14 and iron deposits in pancreatic acinar cells [82]. The regulation of ZIP14 by iron appears to be post-transcriptional because ZIP14 mRNA levels do not vary with iron status [81, 83]. In HepG2 cells, iron deficiency induces plasma membrane ZIP14 endocytosis and degradation via the proteasome [84]. In rat liver and pancreas, the upregulation of ZIP14 levels by iron loading was associated with a downregulation of transferrin receptor 1 levels, implying that these tissues load iron from NTBI rather than TBI [81]. The in vivo role of ZIP14 in NTBI uptake was investigated by using Slc39a14 knockout (Slc39a14-/-) mice [12]. Intravenous administration of 59Fe-labeled ferric citrate revealed that Slc39a14-/- mice took up 70% less 59Fe into the liver and pancreas than did wild-type controls. Moreover, when Slc39a14-/- mice were intercrossed with hemojuvelin knockout Hjv-/- mice, a model of juvenile hemochromatosis, doublemutant Slc39a14-/-; Hjv-/- mice failed to load iron in hepatocytes and pancreatic acinar cells. Similar results were found when Slc39a14-/- mice were intercrossed with hemochromatotic Hfe-/- mice or when they were fed an iron-overloaded diet. Furthermore, when 59Fe was administered via orogastric gavage, Slc39a14-/-; Hjv-/- mice displayed a dramatic impairment in 59Fe uptake by the liver. Collectively, these studies demonstrate that: (i) ZIP14 represents the main pathway for NTBI uptake into the liver and pancreas, (ii) ZIP14 is required for iron loading of hepatocytes and acinar cells, and (iii) ZIP14 is required for efficient hepatic first-past extraction of NTBI from portal blood. The studies additionally showed that diminished NTBI uptake and iron loading of the liver and pancreas of Slc39a14-/- mice was associated with increased NTBI uptake and marked iron accumulation in the kidney and spleen, indicating that these organs have ZIP14-independent mechanisms of NTBI acquisition. Modest increases in heart iron concentrations were also observed in iron-loaded Slc39a14-/- mice although NTBI uptake was not elevated. The fact that ZIP14 is a bona fide in vivo transporter of NTBI, which is not a normal physiological constituent of plasma, can be further understood within the context of its more recently defined physiological role in manganese transport. In 2016, Tuschl et al. [85] reported that individuals with lossof-function mutations in SLC39A14 displayed juvenile-onset parkinsonism-dystonia accompanied by markedly elevated levels of manganese in the blood and brain but not the liver. Slc39a14-/- mice, which recapitulated the altered manganese phenotype, were used to interrogate the physiological role of ZIP14 in manganese homeostasis [86-88]. Radiotracer experiments using orally or intravenously administered

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MnCl2 indicated that ZIP14 is required for manganese uptake by the liver and pancreas [86]. Because manganese homeostasis is maintained mainly by regulating excretion (primarily via the hepatobiliary system but also through pancreatic and intestinal secretions), loss of ZIP14 impairs gastrointestinal excretion of the metal, resulting in manganese accumulation in the blood and extra-hepatopancreatic organs. Hence, under normal physiologic circumstances, ZIP14 in the liver functions to take up manganese (as Mn2+) primarily from portal blood, which carries Mn2+ absorbed from the diet. Under these same conditions, iron in portal blood is likely bound (as Fe3+) to transferrin [20, 89] and is therefore unavailable for transport via ZIP14. However, in iron overload conditions when transferrin is completely saturated, iron will be present as NTBI, thus providing a substrate for ZIP14. Although NTBI is thought to be mostly Fe3+, which is not transported by ZIP14 [80], hepatocytes have an efficient plasma membrane redox system that can convert Fe3+ to Fe2+ [90]. This redox system likely accounts for the fact that perfused liver takes up ferric NTBI complexes as efficiently as ferrous complexes [18]. Data from cell culture experiments using HepG2 cells raised the possibility that ZIP14 additionally participates in the uptake of transferrin-bound iron [91]. This does not seem to be the case in vivo, however, because tissue iron uptake from intravenously administered 59Fe-transferrin is normal in Slc39a14-/- mice [12]. Moreover, Slc39a14-/- mice have normal hemoglobin levels and other hematological parameters, indicating that ZIP14 is dispensable for iron uptake by the erythron, which obtains its iron exclusively via transferrin. A potential role for ZIP14 in NTBI uptake by astrocytes was investigated by using cultured rat brain astrocytes, which were shown to accumulate iron from FeCl3 in a concentration-dependent manner [56]. Cellular iron accumulation was not affected by the presence of equimolar amounts of zinc, and was even enhanced dose-dependently by excess zinc. Given that zinc is a potent inhibitor of ZIP14-mediated iron uptake [92], it seems unlikely that the accumulation of iron from NTBI can be attributed to ZIP14, even though iron uptake per se was not directly measured. Similar to astrocytes, cultured rat primary hippocampal neurons readily take up iron from 59Fe2+-citrate and express ZIP14; however, ZIP14 protein was detectable intracellularly and not at the plasma membrane, suggesting that ZIP14 may not be the primary mediator of NTBI uptake by these neurons [68]. 7.2 ZIP8 (SLC39A8) Among mammalian ZIP proteins, ZIP14 is most homologous to ZIP8. The two proteins are similar in length (491 vs. 459 a.a.’s in human ZIP14 and ZIP8, respectively) and have especially high homology in predicted transmembrane domains, including the putative pore-forming domains IV and V, which are >90% identical [93]. In transmembrane domain V, ZIP14 and ZIP8 are unique in that they have the sequence EEFPH instead of HEXXH found in other ZIPs. This difference in ZIP14 and ZIP8 has been suggested to extend their metal transport capabilities beyond zinc [94]. Transport studies in HEK293T cells and Xenopus oocytes demonstrate that ZIP8 expressed at the plasma membrane transports radiolabeled Fe2+, Zn2+, Mn2+, Co2+, and Cd2+ at pH 7.5 [77]. In rat hepatoma H4IIE cells, levels of total cellular and cell-surface ZIP8 increased with iron loading in a dose-responsive fashion, whereas ZIP8 mRNA levels did not change, suggestive of post-transcriptional regulation by iron. [77]. Despite similarities in structure, substrate profiles, and iron-responsiveness, ZIP8 and ZIP14, display different tissue expression profiles (summarized in [93]), suggesting that they do not function redundantly. In general, ZIP8 is most abundantly expressed in lung, placenta, kidney, and testes, with lower levels of expression in all other tissues examined. Differences in subcellular localization of ZIP14 and ZIP8 further suggest nonredundancy. For example, in the liver, ZIP14 localizes to the basolateral membrane [81] whereas ZIP8 localizes to the apical surface [95]. The investigation of ZIP8’s function in

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vivo has been complicated by the fact that loss of ZIP8 is embryonic lethal. Mice expressing <10% of normal ZIP8 levels (ZIP8 hypomorphic mice) have been described, but the mice die in utero or within 48 hours after birth [96]. The mice exhibited severe iron-deficiency anemia, suggesting that ZIP8 is required for hematopoiesis or placental iron transport. Involvement of ZIP8 in iron transport was demonstrated by impaired uptake of Fe2+ from fetal fibroblasts and liver-derived cells isolated from the hypomorphic mice. Human patients with loss-of-function mutations in ZIP8 (SLC39A8) have been identified, but no data on iron status are provided [97, 98]. The patients did, however, consistently show very low blood manganese levels associated with glycosylation defects, skeletal abnormalities, and severe mental retardation. Recent studies using whole-body inducible Slc39a8 knockout and hepatocyte-specific Slc39a8 knockout mice have concluded that systemic manganese deficiency results from unregulated and excessive biliary Mn losses, with ZIP8 functioning to reclaim the metal from bile [95]. Iron levels in liver, kidney, heart, and brain were normal in the whole-body and liver-specific Slc39a8 knockout mice, suggesting that ZIP8 is dispensable for normal iron metabolism. Studies in iron-loaded Slc39a8 knockout mice will help to determine if ZIP8 participates in NTBI uptake in vivo. Among tissues where ZIP8 is abundantly expressed, the kidney stands out for its ability to readily take up NTBI. Accordingly, ZIP8 has been proposed as a candidate renal NTBI transporter [99, 100], especially since ZIP14 was shown to be dispensable for NTBI uptake by the kidney [12]. Consistent with this possibility is the observation that ZIP8 localizes to the apical membrane of proximal and distal tubules [101]. Moreover, recent studies have found that increased tubular iron deposition in chronic kidney disease is associated with elevated levels of ZIP8 [101]. A role for ZIP8 in NTBI uptake by the retina has also been proposed based on the observation that ZIP8 levels are increased in iron-loaded retinas [102, 103]. In rat primary hippocampal neurons, ZIP8 has been localized to the cell surface, unlike ZIP14 and DMT1, which are primarily intracellular [68]. Suppression of neuronal ZIP8 expression by using shRNA diminished NTBI uptake, suggesting that ZIP8 is a major contributor to NTBI uptake by these cells. 7.3 DMT1 (SLC11A2) DMT1, the first mammalian membrane iron transporter to be identified [104, 105], mediates the transport of a variety of metal ions including Fe2+, Mn2+, Co2+, and Cd2+ [92]. DMT1 is a member of the solute carrier 11 (SLC11) protein family consisting of two members in mammals [106]. Sequence alignment with the prokaryotic homolog Staphylococcus capitis DMT1, whose crystal structure was recently determined [107], indicates that human DMT1 has 12 transmembrane helices with an aqueous cavity and a metal-ion binding site formed between residues in -helices 1 and 6 [108]. DMT1 is well known to play two major roles in iron metabolism: iron absorption by the intestine and iron uptake by developing red blood cells in the bone marrow [109, 110]. In the proximal small intestine, DMT1 resides at the apical membrane of enterocytes where it couples the transport of H+ and Fe2+ into the cell. Dietary iron in the small intestine is often in the Fe3+ state, and therefore a reduction step, carried out at least in part by the apical reductase Dcytb [111], is required prior to uptake by DMT1, which transports Fe2+ but not Fe3+ [92]. In developing erythroid cells of the bone marrow, DMT1 functions in the assimilation of transferrin-bound iron [104]. In the well-known transferrin cycle, monoferric or diferric transferrin binds to transferrin receptor 1 at the cell surface, and the complex is internalized into the endosome, where acidification causes transferrin to release its iron. The liberated Fe3+ is reduced to Fe2+ in the endosome and transported into the cytosol via DMT1. A role for DMT1 in the uptake of NTBI was first investigated by Garrick et al. [112] using reticulocytes from the Belgrade rat, which harbors a disabling mutation in DMT1. Although developing erythroid cells normally obtain their iron exclusively from plasma transferrin, they can take up NTBI [113]. This process

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likely functions to support hemoglobin synthesis (albeit inefficiently) when transferrin is absent, such as in hypotransferrinemic mice. Belgrade rat reticulocytes were found to have an impairment in NTBI uptake, which is also sensitive to inhibitors of endosomal acidification, suggesting that DMT1 can mediate NTBI uptake within the acidified endosome [112]. DMT1 can no doubt transport “non-transferrin-bound iron” (as Fe2+) into the enterocyte and out of the endosome, but can it/does it take up NTBI from the plasma and contribute to tissue iron accumulation? Such a function may seem unlikely given that DMT1 transports iron maximally at pH 5.5 and poorly at 7.5 [105]. The H+-coupled transport of iron allows DMT1 to function well in the acidic microclimates of the intestinal brush border and endosome, but likely not well at a cell surface facing blood plasma with a pH of 7.4. However, functional studies of DMT1 expressed in Xenopus oocytes have demonstrated that the protein can mediate modest H+-uncoupled facilitative Fe2+ transport at pH 7.4, suggesting that DMT1 may take up NTBI at the plasma membrane [114]. Another consideration is whether DMT1 in a particular cell type is expressed at the plasma membrane. DMT1 has four distinct isoforms (DMT1A/IRE(+), DMT1A/IRE(-), DMT1B/IRE(+), and DMT1B/IRE(-)) that may differ in their subcellular distribution, tissue expression, and responsiveness to cellular iron status [115]. In general, the DMT1A/IRE(+) isoform, the one expressed at the apical membrane of enterocytes, localizes to the plasma membrane [116, 117], whereas DMT1B isoforms are mainly located in endosomes or lysosomes [117]. Despite localization of the DMT1A/IRE(+) isoform to the cell surface, the presence of a 3’ IRE in its mRNA would be expected to render the mRNA unstable when cellular iron concentrations increase. Hence, in iron overload conditions, plasma membrane DMT1 levels are predicted to decrease, which would minimize its involvement in NTBI uptake. Immunoblot analysis of DMT1 in rat liver clearly indicates iron-dependent regulation of DMT1, with levels increasing in iron deficiency and decreasing in iron overload [81]. Studies of mice with hepatocyte-specific inactivation of DMT1 formally demonstrated that DMT1 is dispensable for NTBI uptake by the liver [14]. After the intestine, DMT1 is most abundantly expressed in the kidney, suggesting an important function in this organ [105]. Participation of DMT1 in the reabsorption of iron from the glomerular filtrate is implicated by its localization to the apical membrane of proximal and/or distal tubules [101, 118, 119]. Iron can enter the filtrate as NTBI or transferrin-bound iron, since transferrin is filtered, even under normal circumstances [120]. In the acidic pH of the tubular lumen, transferrin likely releases its iron prior to reabsorption [30]. The acidic environment in the tubule would also be conducive for proton-coupled iron transport by DMT1. The role of DMT1 in renal iron handling has been addressed in studies of the Belgrade rat. Veuthy et al. [121] reported that Belgrade rats have elevated urinary iron excretion, but this was associated with renal damage and a 50-fold increase in transferrin in the urine [121]. By contrast, a detailed analysis of food, serum, fecal, and urinary iron levels found that urinary output did not differ between Belgrade rats and controls [122]. Indeed, as the authors of that study noted, the fact that urinary output in Belgrade rats was not increased despite their elevated serum iron levels implies that they actually reabsorbed more iron. Renal iron handling has been additionally linked to DMT1 in a study of the Ca2+ channel blocker nifedipine, which was reported to stimulate DMT1-mediated iron transport, reduce iron accumulation in the kidney and liver, and increase urinary iron excretion in mouse models of iron overload [123]. Whether this effect was mediated through a direct effect of nifedipine on DMT1 function in the kidney was not addressed. Subsequent comprehensive functional analyses of various DMT1 isoforms concluded that nifedipine has no effect on DMT1-mediated iron transport [124]. The possibility that DMT1 participates in NTBI uptake by the heart was initially discounted based on the lack of associations between cellular iron concentrations, DMT1 mRNA levels, and NTBI uptake in cardiomyocytes and fibroblasts [34]. Later studies using mouse or rat heart have confirmed that DMT1

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mRNA levels do not change with iron loading [81, 125, 126], while protein levels either decreased [126] or did not change [81]. On the other hand, two different groups have reported that administration of ebselen, a DMT1 inhibitor [127], mitigated cardiac iron accumulation in iron-loaded mice, suggesting that DMT1 plays a role in NTBI uptake by the heart [128, 129]. Brain interstitial fluid and cerebrospinal fluid are rich in ascorbate [130], implying that a fraction of NTBI in the CNS exists as Fe2+ available for DMT1-mediated uptake. Hence, DMT1’s role in NTBI uptake has been investigated in various brain cell types. In ascorbate-replete cultured rat astrocytes, cellular accumulation of iron from 55Fe-labelled ferric citrate at pH 7.2 was approximately 50% less in the presence of ferristatin, a small molecule inhibitor of DMT1 [131], suggesting that DMT1 mediates NTBI uptake [59]. By contrast, Pelizzoni et al. [70], using primary rat hippocampal astrocytes, concluded that DMT1 likely does not contribute to NTBI uptake by astrocytes because ebselen had no effect on ferrous iron uptake. However, they did find that inflammatory activation of the astrocytes increased DMT1 levels by 15-fold and increased Fe2+ uptake, which could be inhibited by ebselen [60]. It has been proposed that discrepancies among studies of DMT1 in astrocyte NTBI uptake may be influenced by differences in cell culture conditions that can promote different degrees of astrocyte activation [132]. A role for DMT1 in NTBI uptake by neurons was initially suggested by a report of cortical neurons exposed to NMDA [65], but subsequent studies by others concluded otherwise. For example, Pelizzoni et al. [69] found that the DMT1 isoform expressed in primary rat hippocampal neurons (DMT1B/IRE(+)) is readily detectable in the cytosol, but not on the plasma membrane, even when overexpressed. Moreover, overexpression of this isoform failed to increase NTBI uptake in hippocampal neurons, whereas overexpression of the DMT1A isoform, which is not normally expressed in neurons, did. Also using primary rat hippocampal neurons, Ji and Kosman [68] discounted DMT1 in NTBI uptake mainly because kinetic analyses showed that neuronal Fe2+ uptake is competitively inhibited by Zn2+, which is poorly transported by DMT1 [68]. Participation of DMT1 in NTBI uptake by microglia is supported by the observation that LPS treatment of primary adult mouse microglia or a murine microglial cell line increased DMT1 levels and NTBI uptake, and that the increased iron uptake could be blocked by ebselen [73]. The effect of DMT1 in brain iron homeostasis in vivo was recently investigated by using transgenic mice expressing DMT1B/IRE(-) under control of the mouse prion promoter, which overexpressed DMT1 in various neuron-rich regions throughout the brain, including substantia nigra [133]. When mice were fed an iron-supplemented diet (1000 ppm Fe), DMT1 transgenic mice were found to have elevated levels of iron in the substantia nigra, as assessed by Perls’ staining and MRI analysis. Though not directly assessed in this study, the iron accumulation may have resulted from NTBI or TBI uptake, as neurons can acquire iron from both sources [68]. 7.4 LTCCs and TTCCs L-type calcium channels (LTCCs) and T-type calcium channels (TTCCs) are multi-subunit, pore-forming voltage-gated calcium channels (VGCCs) that open in response to membrane depolarization and allow Ca2+ ions to enter cells. In mammals, the VGCC family consists of 10 members, which are divided into three subfamilies: Cav1, Cav2, and Cav3 [134]. The Cav1 subfamily members are more commonly known as LTCCs, and are predominantly expressed in heart, skeletal muscle, neurons, and endocrine cells. The Cav3 subfamily members (TTCCs) have also been reported to be expressed in heart and neurons, as well as kidney and liver. Although VGCCs primarily transport Ca2+, they may also transport other divalent ions including Fe2+ [135-137], Zn2+ [138], and Mn2+ [139]. A role for LTCCs in cardiac NTBI uptake was first suggested by Tsushima et al. [136], who demonstrated that the uptake of 59Fe2+ by isolated rat hearts was diminished by abolishing electrical excitability or by adding the calcium channel blocker nifedipine. Conversely stimulation of LTCCs by Bay K 8644 or

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isoproterenol increased 59Fe2+ uptake. Subsequent in vivo studies reported that treatment of iron-loaded mice with various LTCC blockers (nifedipine, amlodipine, or verapamil) attenuated myocardial iron loading by about 30–50% [129, 140-143]. Consistent with a direct role for LTCCs in cardiac iron loading, transgenic mice overexpressing LTCCs in cardiomyocytes loaded more iron in the heart compared with non-transgenic controls, and as with wild-type mice, the iron loading was attenuated by calcium channel blockers [140]. Although most research on calcium channels has focused on LTCCs as a portal for cardiac iron uptake, TTCCs may also play a role. TTCCs are not usually detectable in normal adult heart, but have been reported to reappear in ventricular hypertrophy [144] and in thalassemic mouse heart [145]. Using isolated cardiomyocytes from thalassemic mice, Kumfu et al. [145] found that efonidipine, a dual TTCC and LTCC blocker, decreased iron uptake whereas the LTCC blocker verapamil had no effect. The ability of efonidipine to decrease cardiac iron accumulation in iron-loaded wild-type or thalassemic mice has been demonstrated in at least 4 studies [129, 143, 146, 147]. In these studies, daily IP administration of efonidipine decreased cardiac iron accumulation by ~30%, similar to the IP administration of the iron chelators desferrioxamine [129], deferasirox, or deferiprone [143]. Administration of the LTCC blockers nifedipine or verapamil equally decreased cardiac iron accumulation [129, 143] but only efonidipine decreased hepatic iron concentrations. Combined administration of desferrioxamine and efonidipine decreased cardiac iron by 50% [147]. Overall, the available data consistently show that treatment of mice with LTCC blockers or dual TTCC and LTCC blockers attenuates cardiac iron accumulation. Formal proof that LTCCs/TTCCs per se mediate cardiac iron uptake, however, would require knockout mouse models. Unfortunately, this is not possible because knockout of the predominant LTCC in the heart (Cav1.2) is lethal, even when induced in adult heart [148, 149]. It should also be noted that while calcium channel blockers attenuate iron loading, they do not completely prevent it, suggesting that alternate iron uptake pathways exist in the myocardium. Indeed, in studies of isolated mouse heart, iron uptake was inhibited by only about 50% in arrested or nifedipine-treated heart, whereas treatment with Cd2+ inhibited 59Fe uptake by ~90% [136]. The identity of this Cd-sensitive iron uptake mechanism in the heart is unknown, but it may be DMT1, ZIP14, or ZIP8, as iron uptake by all of these transporters is potently inhibited by Cd2+. Nonetheless, the success of calcium channel blockade in preclinical animal studies has led to human trials using the LTCC blocker amlodipine to mitigate cardiac iron loading in thalassemia major patients undergoing standard iron chelation therapy. The first trial, an open-label pilot study with 15 participants, found that myocardial iron concentrations, as measured by MRI, decreased by 27% in patients treated with amlodipine (n=5) for 1 year [150]. The second trial was a multicenter, double-blind, randomized, placebo-controlled trial involving 62 patients for 1 year [151]. In subjects starting with above-normal myocardial iron concentrations, those treated with amlodipine had a significant 21% reduction in myocardial iron concentrations, whereas no changes were found in placebo-treated controls. In subjects starting with normal myocardial iron concentrations, no significant changes were observed in either treatment group. Collectively, these data are more consistent with amlodipine increasing myocardial iron losses rather than preventing iron uptake into the heart. Additional trials, including two currently in progress [152], will help to determine whether amlodipine can mitigate myocardial iron accumulation as would be predicted based on the known function of LTCCs in NTBI uptake. It would also be informative to assess iron excretion, especially considering that previous studies reported that the LTCC blocker nifedipine increased urinary iron excretion in mouse models of iron overload [123]. Apart from the heart, VGCCs have been examined as possible mediators of NTBI uptake in neurons, which express numerous VGCCs for various functions, including transcriptional regulation and

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neurotransmitter release [153]. Studies using primary rat hippocampal neurons noted that a mix of VGCC blockers reduced cellular Fe2+ influx by approximately 50% [69]. In neuronal PC12 cells, membrane depolarization by KCl treatment increased Fe2+ influx, which could be inhibited in a dose-responsive fashion by the LTCC blocker nimodipine [67]. A reduction in basal Fe2+ influx by the LTCC blocker nitrendipine has additionally been reported for PC12 cells, but the effect was small (20%) [154]. LTCCs have also been hypothesized to play a role in NTBI uptake in various endocrine cells (e.g., pancreatic beta cells and anterior pituitary gonadotrophs and thyrotrophs) where they are abundantly expressed (summarized in [155]), yet direct evidence for such a role is lacking. Indeed, the conspicuous lack of iron loading in murine beta cells despite their expression of nifedipine-sensitive LTCCs (namely, Cav1.2, the predominant LTCC in the heart) [156] suggests that LTCCs do not mediate NTBI uptake in mouse beta cells.

7.5 TRPC6 TRPC6 is a member of the transient potential receptor (TRP) canonical family of Ca2+-permeable cation channels. A role for TRPC6 in NTBI uptake was initially implicated in studies using PC12 cells. When treated with nerve growth factor, PC12 cells differentiate into a neuronal phenotype and display an increase in NTBI uptake [154], which was associated with de novo expression of TRPC6 [157]. The association was strengthened by the observation that diacylglycerol, an activator of TRPC6, increased NTBI uptake. The ability of TRPC6 to mediate NTBI uptake was shown by using HEK293 cells overexpressing TRPC6 [157]. More recently, a study in primary rat hippocampal astrocytes reported that TRPC blockers had no effect on NTBI uptake [60]. However, treatment of the cells with dihydroxyphenylglycine, a stimulator of TRPC channels, increased NTBI uptake, which could be prevented by TRPC blockers, leading the authors to conclude that TRPCs can provide a route of entry for NTBI in astrocytes [60, 132]. Although TRPC6 was not studied specifically in this study, it is possible that the effect was mediated through TRPC6, as iron transport activity has not been documented in the other six TRPC family members [158]. 8. Cellular uptake of NTBI: the role of ferrireduction Given that plasma NTBI is predominantly Fe3+, while NTBI transporters transport Fe2+, exocytoplasmic ferrireduction is needed prior to cellular Fe3+ uptake. Indeed, this is a strict requirement for DMT1 and ZIP14, which transport iron only as Fe2+ [80, 92]. The dependence of Fe3+ uptake upon reduction to the ferrous state is demonstrated by the use of cell-impermeant Fe2+-specific chelators such as ferrozine or bathophenanthroline disulfonate, which inhibit cellular uptake of iron presented to cells as Fe3+[36, 78, 159]. Cell-surface reduction of iron can be mediated by reductases or reducing agents. A number of mammalian ferrireductases have been identified, including the cytochrome b561 (Cyt b561) family, Steap proteins, prion protein, and alpha-synuclein. Cyt b561 proteins include duodenal Cyt b561 (Dcytb), chromaffin granule Cyt b561, lysosomal Cyt b561, stromal cell-derived receptor 2 (SDR2), and 101F6 [160]. Dcytb, which appears to function as a reductase at the apical membrane of duodenal enterocytes [111], has been detected in other cell types with ferrireductase activity, such as astrocytes, where it has been implicated in NTBI uptake [58, 63]. SDR2 in astrocytes may additionally contribute to the ferrireductase activity of these cells [63]. The Steap family consists of 4 members: Steap1, Steap2, Steap3, and Steap4 [161]. Steap2, Steap3, and Steap4 exhibit ferrireductase activity and increase the uptake of NTBI (as 55Fe-NTA) when overexpressed in HEK293T cells [161, 162]. Steap2 colocalizes with ZIP8 at the cell surface in neurons and has been proposed to mediate ferrireduction prior to ZIP8mediated Fe2+ uptake [68]. Although Steap proteins are detectable at the cell surface and widely

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expressed among tissues [163], their possible participation in NTBI uptake in vivo has yet to be examined. An in vivo role of prion protein (PrP), a ubiquitously expressed cell surface glycoprotein with ferrireductase activity [164], is suggested by studies of PrP knockout (PrP-/-) mice [164]. After intraperitoneal administration of 59Fe-labeled ferric citrate, PrP-/- mice took up approximately 25% less 59 Fe into the liver and pancreas [165] and kidney [166] when compared with PrP+/+ controls. Diminished uptake of 59Fe by PrP-/- kidney was also demonstrated after intravenous administration of 59Fe-labeled ferric citrate [166]. In HepG2 cells, co-expression of PrP with ZIP8, ZIP14, and DMT1 increased iron loading from ferric ammonium citrate, suggesting that PrP-mediated reduction functions cooperatively with these NTBI transporters [165]. Reductase activity for -synuclein, the protein implicated in the pathogenesis of Parkinson disease and other neurological diseases, has been demonstrated by using purified recombinant human -synuclein and neuronal cell lines overexpressing the protein [167]. An in vivo role of -synuclein is supported by a recent study showing that AAV-mediated overexpression of human -synuclein in nigral dopaminergic neurons in rat brain increased ferrireductase activity in the substantia nigra [168]. In the brain, non-enzymatic ferrireduction via ascorbate seems likely given that ascorbate concentrations are ~400 M in the extracellular fluid and millimolar concentrations within cells [169]. Consistent with this possibility is a study of primary cultures of ascorbate-replete rat astrocytes showing that incubation of the cells with the ascorbate-oxidizing enzyme, ascorbate oxidase, abolished the reduction of ferric citrate and decreased iron accumulation by the cells [59]. It is also possible that ascorbate participates in extracellular ferrireduction for NTBI uptake by peripheral tissues, as plasma ascorbate concentrations in unsupplemented individuals are 20–60 M [170].

9. Remaining research questions in NTBI transport Since the first demonstration 15 years ago that LTCCs mediated cardiac NTBI uptake in vivo [140], significant progress has been made in the identification and characterization of additional NTBI transporters that play important roles in NTBI uptake by other tissues and cells (Figure 1). Nonetheless, important questions remain. For example, how does the anterior pituitary take up NTBI and accumulate iron in iron overload? This question is of high clinical significance, as iron deposition in the pituitary is associated with endocrine disorders such as hypogonadotropic hypogonadism, which remains the most common complication in patients with thalassemia major. How do cardiomyocytes take up NTBI? LTCCs/TTCCs are important contributors no doubt, yet they account for only about half of total NTBI uptake by the heart. The identification of other NTBI mechanisms in cardiomyocytes may lead to additional complementary approaches that could further reduce the risk of iron overload-related cardiomyopathy, the leading cause of death in patients with thalassemia major. This is of high clinical significance because even small reductions in myocardial iron concentrations are associated with improved clinical outcomes. How does the kidney reabsorb NTBI from the glomerular filtrate? The observation that DMT1, ZIP14, and ZIP8 all localize to kidney tubules is intriguing and may be suggestive of redundancy in renal iron reabsorption mechanisms. Identification of these mechanisms could lead to therapeutic inhibitors that would increase urinary iron excretion and lessen body iron burden. How is NTBI taken up by cells of the CNS? While NTBI in the plasma is generally pathological, NTBI in the CNS appears to be physiological and the exclusive source of iron for astrocytes in vivo. However, how these cells and others of the CNS take up NTBI is poorly understood. It is also critical to understand how NTBI transport in the CNS is affected by neuroinflammation, as an iron-dependent regulated form of cell death (ferroptosis) is emerging as a major contributor to the pathophysiology of neurological disorders such as Parkinson disease and Alzheimer’s disease [171, 172].

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Acknowledgments This work was supported by National Institutes of Health grant DK080706 (to M.D.K.).

Conflict of interest The author has no conflict of interest to declare.

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Figures:

Fig. 1. Mechanisms of NTBI uptake by organs and main cell types that take up NTBI and/or accumulate iron in iron overload. Most NTBI in plasma is rapidly and efficiently cleared by the liver via ZIP14 in hepatocytes. ZIP14 in the pancreas, mainly in acinar cells, contributes to NTBI clearance and assumes a greater role as the liver approaches its iron storage capacity. Iron loading of the pancreas is paralleled by a

24

slower accumulation of iron in the heart, which takes up NTBI via LTCCs/TTCCs and other unknown mechanisms (indicated by a question mark). The kidney takes up NTBI as efficiently as the pancreas, yet which transporters are involved remains unresolved. In the CNS, neurons likely utilize ZIP8 to take up NTBI from brain interstitial fluid or cerebrospinal fluid. Mechanisms of NTBI uptake by astrocytes and microglia are poorly defined; however, after inflammatory activation, DMT1 plays a role. How endocrine glands (anterior pituitary, thyroid, and adrenal) take up NTBI is unknown.

Table 1 Non-transferrin-bound iron transporters and evidence supporting their role in NTBI uptake. Protein Gene First Evidence type implication in NTBI transport (year) In vivo Cell culture inhibition transgenic knockout 1 [78] (2006) [78] [80] [43] [12] SLC39A14 ZIP14 [136] (1999) [136] [154] [140] [142] [140] LTCC Cav1.2/1.3 [140] [67] [69]

TTCC

Cav3.1

[145] (2011)

[145]

DMT1

SLC11A2

[112] (1999)

[112] [65] [59]

ZIP8 TRPC6

SLC39A8 TRPC6

[77] (2012) [157] (2004)

[145] [143] [150] [151] [129] [143] [146] [147] [125] [126]

-

-

[133]

-

-

-

-

[60] [73] [77] [96] [68] [157][60]

1

Abbreviations: ZIP14, ZRT/IRT-like protein 14; LTCC, L-type calcium channel; TTCC, T-type calcium channel; DMT1, divalent metal-ion transporter-1; ZIP8, ZRT/IRT-like protein 8; TRPC6, transient receptor potential cation channel subfamily C member 6.

Highlights     

Non-transferrin-bound iron (NTBI) appears in the plasma in iron overload. NTBI is a major contributor to pathological iron loading of various tissues. Tissue uptake of NTBI is mediated by plasma membrane transporters. NTBI transporters include ZIP14, LTCCs, TTCCs, DMT1, ZIP8, and TRPC6. NTBI transporters function in a cell-type and tissue-specific manner.