Zinc is involved in a variety of biological processes, as a structural, catalytic, and intracellular and intercellular signaling component. Thus zinc homeostasis is tightly controlled at the whole body, tissue, cellular, and subcellular levels by a number of proteins, with zinc transporters being particularly important. In metazoan, two zinc transporter families, Zn transporters (ZnT) and Zrt-, Irt-related proteins (ZIP) function in zinc mobilization of influx, efflux, and compartmentalization/ sequestration across biological membranes. During the last two decades, significant progress has been made in understanding the molecular properties, expression, regulation, and cellular and physiological roles of ZnT and ZIP transporters, which underpin the multifarious functions of zinc. Moreover, growing evidence indicates that malfunctioning zinc homeostasis due to zinc transporter dysfunction results in the onset and progression of a variety of diseases. This review summarizes current progress in our understanding of each ZnT and ZIP transporter from the perspective of zinc physiology and pathogenesis, discussing challenging issues in their structure and zinc transport mechanisms.

Zinc concentrations in breast milk are considerably higher than those of the maternal serum, to meet the infant's requirements for normal growth and development. Thus, effective mechanisms ensuring secretion of large amounts of zinc into the milk operate in mammary epithelial cells during lactation. ZnT2 was recently found to play an essential role in the secretion of zinc into milk. Heterozygous mutations of human ZnT2 (hZnT2), including H54R and G87R, in mothers result in low (>75% reduction) secretion of zinc into the breast milk, and infants fed on the milk develop transient neonatal zinc deficiency. We identified two novel missense mutations in the SLC30A2/ZnT2 gene in a Japanese mother with low milk zinc concentrations (>90% reduction) whose infant developed severe zinc deficiency; a T to C transition (c.454T>C) at exon 4, which substitutes a tryptophan residue with an arginine residue (W152R), and a C to T transition (c.887C>T) at exon 7, which substitutes a serine residue with a leucine residue (S296L). Biochemical characterization using zinc-sensitive DT40 cells indicated that the W152R mutation abolished the abilities to transport zinc and to form a dimer complex, indicating a loss-of-function mutation. The S296L mutation retained both abilities but was extremely destabilized. The two mutations were found on different alleles, indicating that the genotype of the mother with low milk zinc was compound heterozygous. These results show novel compound heterozygous mutations in the SLC30A2/ZnT2 gene causing zinc deficiency in a breast-fed infant.

Our results indicate that many SLC30A2/ZnT2 mutations cause or potentially cause TNZD. Genetic information concerning TNZD pathogenesis is limited, and our results suggest that the TNZD frequency may be higher than previously thought.

The activation process of secretory or membrane-bound zinc enzymes is thought to be a highly coordinated process involving zinc transport, trafficking, transfer and coordination. We have previously shown that secretory and membrane-bound zinc enzymes are activated in the early secretory pathway (ESP) via zinc-loading by the zinc transporter 5 (ZnT5)-ZnT6 hetero-complex and ZnT7 homo-complex (zinc transport complexes). However, how other proteins conducting zinc metabolism affect the activation of these enzymes remains unknown. Here, we investigated this issue by disruption and re-expression of genes known to be involved in cytoplasmic zinc metabolism, using a zinc enzyme, tissue non-specific alkaline phosphatase (TNAP), as a reporter. We found that TNAP activity was significantly reduced in cells deficient in ZnT1, Metallothionein (MT) and ZnT4 genes (ZnT1 −/− MT −/− ZnT4 −/− cells), in spite of increased cytosolic zinc levels. The reduced TNAP activity in ZnT1 −/− MT −/− ZnT4 −/− cells was not restored when cytosolic zinc levels were normalized to levels comparable with those of wild-type cells, but was reversely restored by extreme zinc supplementation via zinc-loading by the zinc transport complexes. Moreover, the reduced TNAP activity was adequately restored by re-expression of mammalian counterparts of ZnT1, MT and ZnT4, but not by zinc transport-incompetent mutants of ZnT1 and ZnT4. In ZnT1 −/− MT −/− ZnT4 −/− cells, the secretory pathway normally operates. These findings suggest that cooperative zinc handling of ZnT1, MT and ZnT4 in the cytoplasm is required for full activation of TNAP in the ESP, and present clear evidence that the activation process of zinc enzymes is elaborately controlled.

A gradually increasing number of transient neonatal zinc deficiency (TNZD) cases was recently reported, all of which were associated with inactivating ZnT2 mutations. Here we characterized the impact of three novel heterozygous ZnT2 mutations G280R, T312M, and E355Q, which cause TNZD in exclusively breastfed infants of Japanese mothers. We used the bimolecular fluorescence complementation (BiFC) assay to provide direct visual evidence for the in situ dimerization of these ZnT2 mutants, and to explore their subcellular localization. Moreover, using three complementary functional assays, zinc accumulation using BiFC-Zinquin and Zinpyr-1 fluorescence as well as zinc toxicity assay, we determined the impact of these ZnT2 mutations on vesicular zinc accumulation. Although all three mutants formed homodimers with the wild type (WT) ZnT2 and retained substantial vesicular localization, as well as vesicular zinc accumulation, they had no dominant-negative effect over the WT ZnT2. Furthermore, using advanced bioinformatics, structural modeling, and site-directed mutagenesis we found that these mutations localized at key residues, which play an important physiological role in zinc coordination (G280R and E355Q) and zinc permeation (T312M). Collectively, our findings establish that some heterozygous loss of function ZnT2 mutations disrupt zinc binding and zinc permeation, thereby suggesting a haploinsufficiency state for the unaffected WT ZnT2 allele in TNZD pathogenesis. These results highlight the burning need for the development of a suitable genetic screen for the early diagnosis of TNZD to prevent morbidity.In the past 50 years, the key role of zinc in human health was established (1). Zinc is crucial for central physiological processes including DNA and protein synthesis, enzyme activity, intracellular signaling, immune function, fertility, as well as growth and development (1-3). Zinc homeostasis is primarily regulated by two zinc transporter families as well as by zincbinding proteins. The SLC39A gene family encodes for ZIP1-14 transporters, which are responsible for zinc uptake into the cytosol from the extracellular fluid or from intracellular vesicles (4). In contrast, the SLC30A gene family encodes for the transporters ZnT1-10, which are responsible for zinc secretion from the cytosol to intracellular organelles or through the plasma membrane outside the cells (2, 4).The recent position of the Academy of Nutrition and Dietetics (5) is that breastfeeding in the first 6 months of life can provide adequate micronutrients and macronutrients for optimal growth and development. This statement is correct for the vast majority of infants around the world. However, recent reports indicate that some exclusively breastfed infants suffer from severe zinc deficiency due to lack of zinc in the breast milk they consume (6 -11). These cases were referred to as transient neonatal zinc deficiency (TNZD), 4 as the symptoms can be treated with zinc supplementation to the diet of the infant (12, 13). In contrast to TNZD, which appe...