Restraint stress up-regulates expression of zinc transporter Zip14 mRNA in mouse liver

The zinc concentration in the liver was significantly higher in mice at 12 h after the onset of restraint stress than that without stress. The IL-6 protein level in the serum was transiently elevated at 3 h after the onset of restraint stress, and the IL-6-responsive zinc transporter Zip14 mRNA in the liver was expressed markedly at 6 h. These results suggest that Zip14 plays a major role in the mechanism responsible for accumulation of zinc in the liver under restraint stress.     

The mammalian Zip5 protein is a zinc transporter that localizes to the basolateral surface of polarized cells

The mouse and human Zip5 proteins are members of the ZIP family of metal ion transporters. In this study, we present evidence that mouse Zip5 is a zinc uptake transporter that is specific for Zn(II) over other potential metal ion substrates. We also show that, unlike many other mammalian ZIP proteins, the endocytic removal of mZip5 from the plasma membrane is not triggered by zinc treatment. Thus, the activity of mZip5 does not appear to be down-regulated by zinc repletion. Zip5 expression is restricted to many tissues important for zinc homeostasis, including the intestine, pancreas, liver, and kidney. Zip5 is similar in sequence to the Zip4 protein, which is involved in the uptake of dietary zinc. Co-expression of Zip4 and Zip5 in the intestine led to the hypothesis that these proteins play overlapping roles in the uptake of dietary zinc across the apical membrane of intestinal enterocytes. Surprisingly, however, we found that mZip5 localizes specifically to the basolateral membrane of polarized Madin-Darby canine kidney cells. These observations suggest that Zip5 plays a novel role in polarized cells by carrying out serosal-to-mucosal zinc transport. Furthermore, given its expression in tissues important to zinc homeostasis, we propose that Zip5 plays a central role in controlling organismal zinc status.     

Functional properties and cellular distribution of the system A glutamine transporter SNAT1 support specialized roles in central neurons

Glutamine, the preferred precursor for neurotransmitter glutamate and GABA, is likely to be the principal substrate for the neuronal System A transporter SNAT1 in vivo. We explored the functional properties of SNAT1 (the product of the rat Slc38a1 gene) by measuring radiotracer uptake and currents associated with SNAT1 expression in Xenopus oocytes and determined the neuronal-phenotypic and cellular distribution of SNAT1 by confocal laser-scanning microscopy alongside other markers. We found that SNAT1 mediates transport of small, neutral, aliphatic amino acids including glutamine (K0.5 approximately 0.3 mm), alanine, and the System A-specific analogue 2-(methylamino)isobutyrate. Amino acid transport is driven by the Na+ electrochemical gradient. The voltage-dependent binding of Na+ precedes that of the amino acid in a simultaneous transport mechanism. Li+ (but not H+) can substitute for Na+ but results in reduced Vmax. In the absence of amino acid, SNAT1 mediates Na+-dependent presteady-state currents (Qmax approximately 9 nC) and a nonsaturable cation leak with selectivity Na+, Li+ > H+, K+. Simultaneous flux and current measurements indicate coupling stoichiometry of 1 Na+ per 1 amino acid. SNAT1 protein was detected in somata and proximal dendrites but not nerve terminals of glutamatergic and GABAergic neurons throughout the adult CNS. We did not detect SNAT1 expression in astrocytes but detected its expression on the luminal membranes of the ependyma. The functional properties and cellular distribution of SNAT1 support a primary role for SNAT1 in glutamine transport serving the glutamate/GABA-glutamine cycle in central neurons. Localization of SNAT1 to certain dopaminergic neurons of the substantia nigra and cholinergic motoneurons suggests that SNAT1 may play additional specialized roles, providing metabolic fuel (via alpha-ketoglutarate) or precursors (cysteine, glycine) for glutathione synthesis.     

Zip3 plays a major role in zinc uptake into mammary epithelial cells and is regulated by prolactin

During lactation, a substantial amount of Zn²⁺ is transferred by the mammary gland from the maternal circulation into milk; thus secretory mammary epithelial cells must tightly regulate Zn²⁺ transport to ensure optimal Zn²⁺ transfer to the suckling neonate. To date, six Zn²⁺ import proteins (Zip1–6) have been identified; however, Zip3 expression is restricted to tissues with unique requirements for Zn²⁺, such as the mammary gland, which suggests that it may play a specialized role in this tissue. In the present study, we have used a unique mammary epithelial cell model (HC11) to characterize the role of Zip3 in mammary epithelial cell Zn²⁺ transport. Confocal microscopy demonstrated that Zip3 is localized to the cell surface in mammary epithelial cells and transiently relocalized to an intracellular compartment in cells with a secretory phenotype. Total ⁶⁵Zn transport was higher in secreting cells, while gene silencing of Zip3 decreased ⁶⁵Zn uptake into mammary epithelial cells, particularly in those with a secretory phenotype. Finally, reduced expression of Zip3 ultimately resulted in cell death, indicating that mammary epithelial cells have a unique requirement for Zip3-mediated Zn²⁺ import, which may reflect the unique requirement for Zn²⁺ of this highly specialized cell type and thus provides a physiological explanation for the restricted tissue distribution of this Zn²⁺ importer. zinc transport; mammary gland; lactation     

Human ZIP1 is a major zinc uptake transporter for the accumulation of zinc in prostate cells

The prostate gland of humans and other animals accumulates a level of zinc that is 3-10 times greater than that found in other tissues. Associated with this ability to accumulate zinc is a rapid zinc uptake process in human prostate cells, which we previously identified as the hZIP1 zinc transporter. We now provide additional evidence that hZIP1 is an important operational transporter that allows for the transport and accumulation of zinc. The studies reveal that hZIP1 (SLC39A1) but not hZIP2 (SLC39A2) is expressed in the zinc-accumulating human prostate cell lines, LNCaP and PC-3. Transfected PC-3 cells that overexpress hZIP1 exhibit increased uptake and accumulation of zinc. The V(max) for zinc uptake was increased with no change in K(m). Along with the increased intracellular accumulation of zinc, the overexpression of hZIP1 also results in the inhibition of growth of PC-3 cells. Down-regulation of hZIP1 by treatment of PC-3 cells with hZIP1 antisense oligonucleotide resulted in a decreased zinc uptake. Uptake of zinc from zinc chelated with citrate was as rapid as from free zinc ions; however, the cells did not take up zinc chelated with EDTA. The cellular uptake of zinc is not dependent upon an available pool of free Zn(2+) ions. Instead, the mechanism of transport appears to involve the transport of zinc from low molecular weight ligands that exist in circulation as relatively loosely bound complexes with zinc.     

LiZIP3 is a cellular zinc transporter that mediates the tightly regulated import of zinc in Leishmania infantum parasites

Cellular zinc homeostasis ensures that the intracellular concentration of this element is kept within limits that enable its participation in critical physiological processes without exerting toxic effects. We report here the identification and characterization of the first mediator of zinc homeostasis in Leishmania infantum, LiZIP3, a member of the ZIP family of divalent metal‐ion transporters. The zinc transporter activity of LiZIP3 was first disclosed by its capacity to rescue the growth of Saccharomyces cerevisiae strains deficient in zinc acquisition. Subsequent expression of LiZIP3 in Xenopus laevis oocytes was shown to stimulate the uptake of a broad range of metal ions, among which Zn²⁺ was the preferred LiZIP3 substrate (K_0.5 ≈ 0.1 μM). Evidence that LiZIP3 functions as a zinc importer in L. infantum came from the observations that the protein locates to the cell membrane and that its overexpression leads to augmented zinc internalization. Importantly, expression and cell‐surface location of LiZIP3 are lost when parasites face high zinc bioavailability. LiZIP3 decline in response to zinc is regulated at the mRNA level in a process involving (a) short‐lived protein(s). Collectively, our data reveal that LiZIP3 enables L. infantum to acquire zinc in a highly regulated manner, hence contributing to zinc homeostasis.     

ZnT5 variant B is a bidirectional zinc transporter and mediates zinc uptake in human intestinal Caco-2 cells

Zinc is an essential micronutrient, so it is important to elucidate the molecular mechanisms of zinc homeostasis, including the functional properties of zinc transporters. Mammalian zinc transporters are classified in two major families: the SLC30 (ZnT) family and the SLC39 family. The prevailing view is that SLC30 family transporters function to reduce cytosolic zinc concentration, either through efflux across the plasma membrane or through sequestration in intracellular compartments, and that SLC39 family transporters function in the opposite direction to increase cytosolic zinc concentration. We demonstrated that human ZnT5 variant B (ZnT5B (hZTL1)), an isoform expressed at the plasma membrane, operates in both the uptake and the efflux directions when expressed in Xenopus laevis oocytes. We measured increased activity of the zinc-responsive metallothionein 2a (MT2a) promoter when ZnT5b was co-expressed with an MT2a promoter-reporter plasmid construct in human intestinal Caco-2 cells, indicating increased total intracellular zinc concentration. Increased cytoplasmic zinc concentration mediated by ZnT5B, in the absence of effects on intracellular zinc sequestration by the Golgi apparatus or endoplasmic reticulum, was also confirmed by a dramatically enhanced signal from the zinc fluorophore Rhodzin-3 throughout the cytoplasm of Caco-2 cells overexpressing ZnT5B at the plasma membrane when compared with control cells. Our findings demonstrate clearly that, in addition to mediating zinc efflux, ZnT5B at the plasma membrane can function to increase cytoplasmic zinc concentration, thus indicating a need to reevaluate the current paradigm that SLC30 family zinc transporters operate exclusively to decrease cytosolic zinc concentration.     

The hereditary hemochromatosis protein, HFE, inhibits iron uptake via down-regulation of Zip14 in HepG2 cells

Lack of functional hereditary hemochromatosis protein, HFE, causes iron overload predominantly in hepatocytes, the major site of HFE expression in the liver. In this study, we investigated the role of HFE in the regulation of both transferrin-bound iron (TBI) and non-transferrin-bound iron (NTBI) uptake in HepG2 cells, a human hepatoma cell line. Expression of HFE decreased both TBI and NTBI uptake. It also resulted in a decrease in the protein levels of Zip14 with no evident change in the mRNA level of Zip14. Zip14 (Slc39a14) is a metal transporter that mediates NTBI into cells (Liuzzi, J. P., Aydemir, F., Nam, H., Knutson, M. D., and Cousins, R. J. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 13612-13617). Knockdown of Zip14 with siRNA abolished the effect of HFE on NTBI uptake. To determine if HFE had a similar effect on Zip14 in another cell line, HeLa cells expressing HFE under the tetracycline-repressible promoter were transfected with Zip14. As in HepG2 cells, HFE expression inhibited NTBI uptake by approximately 50% and decreased Zip14 protein levels. Further analysis of protein turnover indicated that the half-life of Zip14 is lower in cells that express HFE. These results suggest that HFE decreases the stability of Zip14 and therefore reduces the iron loading in HepG2 cells.     

Cloning and functional characterization of a mammalian zinc transporter that confers resistance to zinc.

A cDNA encoding a zinc transporter (ZnT-1) was isolated from a rat kidney cDNA expression library by complementation of a mutated, zinc-sensitive BHK cell line. This cDNA was used to isolate the homologous mouse ZnT-1 gene. The proteins predicted for these transporters contain six membrane-spanning domains, a large intracellular loop and a C-terminal tail. ZnT-1 is homologous to zinc and cobalt resistance genes of yeast. Immunocytochemistry with an antibody to a myc epitope added to the C-terminus of ZnT-1 revealed localization to the plasma membrane. Transformation of normal cells with a mutant ZnT-1 lacking the first membrane-spanning domain conferred zinc sensitivity on wild-type cells, suggesting that ZnT-1 functions as a multimer. Deletion of the first two membrane-spanning domains resulted in a non-functional molecule, whereas deletion of the C-terminal tail produced a toxic phenotype. Mutant cells have a slightly higher steady-state level of intracellular zinc and high basal expression of a zinc-dependent reporter gene compared with normal cells. Mutant cells have a lower turnover of 65Zn compared with normal cells or mutant cells transformed with ZnT-1. We propose that ZnT-1 transports zinc out of cells and that its absence accounts for the increased sensitivity of mutant cells to zinc toxicity.     

Evidence for a zinc uptake transporter in human prostate cancer cells which is regulated by prolactin and testosterone.

The glandular epithelial cells of the human prostate gland have the unique capability and function of accumulating the highest zinc levels of any soft tissue in the body. Zinc accumulation in the prostate is regulated by prolactin and testosterone; however, little information is available concerning the mechanisms associated with zinc accumulation and its regulation in prostate epithelial cells. In the present studies the uptake and accumulation of zinc were determined in the human malignant prostate cell lines LNCaP and PC-3. The results demonstrate that LNCaP cells and PC-3 cells possess the unique capability of accumulating high levels of zinc. Zinc accumulation in both cell types is stimulated by physiological concentrations of prolactin and testosterone. The studies reveal that these cells contain a rapid zinc uptake process indicative of a plasma membrane zinc transporter. Initial kinetic studies demonstrate that the rapid uptake of zinc is effective under physiological conditions that reflect the total and mobile zinc levels in circulation. Correspondingly, genetic studies demonstrate the expression of a ZIP family zinc uptake transporter in both LNCaP and PC-3 cells. The rapid zinc uptake transport process is stimulated by treatment of cells with physiological levels of prolactin and testosterone, which possibly is the result of the regulation of the ZIP-type zinc transporter gene. These zinc-accumulating characteristics are specific for prostate cells. The studies support the concept that these prostate cells express a unique hormone-responsive, plasma membrane-associated, rapid zinc uptake transporter gene associated with their unique ability to accumulate high zinc levels.     

Arabidopsis LHT1 is a high-affinity transporter for cellular amino acid uptake in both root epidermis and leaf mesophyll

Amino acid transport in plants is mediated by at least two large families of plasma membrane transporters. Arabidopsis thaliana, a nonmycorrhizal species, is able to grow on media containing amino acids as the sole nitrogen source. Arabidopsis amino acid permease (AAP) subfamily genes are preferentially expressed in the vascular tissue, suggesting roles in long-distance transport between organs. We show that the broad-specificity, high-affinity amino acid transporter LYSINE HISTIDINE TRANSPORTER1 (LHT1), an AAP homolog, is expressed in both the rhizodermis and mesophyll of Arabidopsis. Seedlings deficient in LHT1 cannot use Glu or Asp as sole nitrogen sources because of the severe inhibition of amino acid uptake from the medium, and uptake of amino acids into mesophyll protoplasts is inhibited. Interestingly, lht1 mutants, which show growth defects on fertilized soil, can be rescued when LHT1 is reexpressed in green tissue. These findings are consistent with two major LHT1 functions: uptake in roots and supply of leaf mesophyll with xylem-derived amino acids. The capacity for amino acid uptake, and thus nitrogen use efficiency under limited inorganic N supply, is increased severalfold by LHT1 overexpression. These results suggest that LHT1 overexpression may improve the N efficiency of plant growth under limiting nitrogen, and the mutant analyses may enhance our understanding of N cycling in plants.     

Generation and characterization of mice lacking the zinc uptake transporter ZIP3

The mouse ZIP3 (SLC39A3) gene encodes an eight-transmembrane-domain protein that has been conserved in mammals and can function to transport zinc. To analyze the expression of ZIP3 in the early embryo and neonate and to determine its in vivo function, we generated ZIP3 null mice in which the ZIP3 open reading frame was replaced with that of the enhanced green fluorescent protein (EGFP) reporter. EGFP fluorescence revealed that ZIP3 was expressed in the inner cell mass of the blastocyst and later during embryonic development in many tissues. Elevated expression was apparent in the embryonic brain and neurotube and neonatal gonads. Homozygous knockout mice were viable and fertile and under normal growth conditions exhibited no obvious phenotypic abnormalities. Deletion of ZIP3 did not alter zinc homeostasis at the molecular level as assessed by essential metal levels and the expression of zinc-responsive genes. In knockout mice stressed with a zinc-deficient diet during pregnancy or at weaning, a subtle increase in the sensitivity to abnormal morphogenesis of the embryo and to depletion of thymic pre-T cells, respectively, was noted. These results suggest that this protein plays an ancillary role in zinc homeostasis in mice.     

Role of the iron transporter OsNRAMP1 in cadmium uptake and accumulation in rice

The heavy metal cadmium (Cd) is toxic to humans, and its accumulation in rice grains is a major agricultural problem. Rice has seven putative metal transporter NRAMP genes, but microarray analysis showed that only OsNRAMP1 is highly up-regulated by iron (Fe) deficiency. OsNRAMP1 localized to the plasma membrane and transported Cd as well as Fe. OsNRAMP1 expression was observed mainly in roots and was higher in the roots of a high-Cd-accumulating cultivar (Habataki) than in those of a low-Cd-accumulating cultivar (Sasanishiki). The amino acid sequence of OsNRAMP1 in the Sasanishiki and Habataki cultivars was found to be 100% identical. These results suggest that OsNRAMP1 participates in cellular Cd uptake and that the differences observed in Cd accumulation among cultivars are because of differences in OsNRAMP1 expression levels in roots.     

The cellular and subcellular localization of zinc transporter 7 in the mouse spinal cord

The present work addresses the cellular and subcellular localization of the zinc transporter 7 (ZNT7, SLC30a7) protein and the distribution of zinc ions (Zn2+) in the mouse spinal cord. Our results indicated that the ZNT7 immunoreactive neurons were widely distributed in the Rexed's laminae of the gray matter in all spinal segments examined. The ependyma cells of the central canal and glia cells in the white matter were also shown ZNT7-positive. The ZNT7 immunoreactivity was mainly detected in the perinuclear regions of ZNT7-positive cells in the spinal gray matter. For ependyma cells, the immunoreactivity of ZNT7 was detected in the cytoplasm near the lumina of the central canal. Ultrastructural localization showed that ZNT7 was predominately present in the membrane of the Golgi stacks. The double immunofluorescence studies confirmed this result. Other intracellular organelles including the endoplasmic reticulum, mitochondria and lysosomes were devoid of ZNT7-immunostaining. The chelatable Zn2+ ions in the spinal cord were found predominantly in the terminals of the neuron rather than the cell body in the gray matter. However, overlapping distribution of chelatable Zn2+ ions and ZNT7 was found in the ependyma cells. The present study supports the notion that ZNT7 may function to supply zinc ions to the newly synthesized metalloproteins in the secretory pathway of the spinal neuron and the ependyma cell.