The Journal of Nutrition Vol. 128 No. 4 April 1998, pp. 667-670

Mammalian Zinc Transporters^1,2

Robert J. McMahon and Robert J. Cousins³

Center for Nutritional Sciences and Food Science and Human Nutrition Department, University of Florida, Gainesville, FL 32611

	ABSTRACT

Abstract Introduction References

Genes that are involved in mammalian zinc transport recently have been cloned. These all predict proteins with multiple membrane spanning regions, and most have a histidine-rich intracellular loop. ZnT-1 was the first cloned and is associated with zinc efflux. It is found in all tissues examined, and, at least in some, ZnT-1 expression is regulated by dietary zinc intake. In enterocytes of the small intestine and renal tubular cells, ZnT-1 is localized to the basolateral membrane, suggesting an orientation that is consistent with zinc absorption/retention. ZnT-2 is also an exporter and may be involved in zinc efflux or uptake into vesicles in intestine, kidney, and testis. ZnT-3 is involved in zinc uptake into vesicles in neurons and possibly in testis. ZnT-4 is also an exporter and is highly expressed in mammary gland and brain. The divalent cation transporter 1 (DCT1) is regulated by iron, but exhibits transport activity for a number of trace elements including zinc. Description of a family of zinc transporters bridges the integrative and reductionist approach to the study of zinc metabolism. Other members of this transporter family may emerge. Many of these may be regulated by zinc, and some may respond to immune challenge, oxidative stress, and competing metals in the dietary supply. Collectively, description of transporters that influence cellular zinc uptake and efflux will provide a clearer understanding of the molecular events that regulate zinc absorption and homeostasis.

	INTRODUCTION

Abstract Introduction References

Zinc is a small, hydrophilic, highly charged species which cannot cross biological membranes by passive diffusion. Therefore, specialized mechanisms are required for both its uptake and release (da Silva and Williams 1991). The kinetic characteristics of this process have been well described in many systems, including intact animals, isolated cells from various sources, and intestinal segments (reviewed in Reyes 1996).

At a reductionist level, the transport of zinc has been studied in several different isolated cell types, including hepatocytes (Failla and Cousins 1978, Pattison and Cousins 1986, Taylor and Simons 1994), placental trophoblasts (Mas and Sarkar 1991), fibroblasts (Ackland et al. 1988), intestinal cells (Raffaniello et al. 1992), and endothelial cells (Bobilya et al. 1992) as well as membrane vesicles (Menard and Cousins 1983, Oestreicher and Cousins 1989, Tacnet et al. 1990, 1993). Although differences exist in zinc transport in these systems, all exhibit some common characteristics. Zinc transport is a time-, temperature-, and pH-sensitive process, and there appears to be both saturable and nonsaturable components (Reyes 1996). There also may be an energy requirement for some aspects of zinc transport. It is probable that zinc can be transported across membranes as a free ion (Reyes 1996); however, some evidence demonstrates that the presence of zinc-binding ligands can significantly affect zinc transport into cells (Cousins 1996). The mechanism of zinc efflux from cells has received much less study. It must be emphasized that zinc accumulation by cells, a sum of influx and efflux processes, rather than true membrane transport is actually what usually is studied experimentally.

Despite the solid work describing the kinetics and characteristics of zinc transport and the efflux process, no proteins directly associated with zinc transport had been described until recently. In this review, which is about the field of mammalian zinc transporters in its infancy, we will address briefly what is known about each of these transporters, compare their structure and expression, and address the nutritionally relevant importance these genes might play in zinc metabolism. Brevity prevents discussion of the plethora of research on bacterial and yeast zinc transporters and zinc resistance genes (Eide 1997) that have provided information relevant to mammalian homologues.

ZNT-1 Transporter.  The first zinc transporter was described by Palmiter and Findley (1995) and termed ZnT-1. When transfected into zinc sensitive baby hamster kidney (BHK)⁴ cells, the ZnT-1 gene conferred the ability to resist high levels of extracellular zinc (Palmiter and Findley 1995). The rat cDNA sequence of this gene predicts a protein of 507 amino acids with six putative membrane spanning regions and, following the "inside positive rule" (Von Heijne 1994), the amino and carboxy terminus would reside intracellularly. A ZnT/lacZ fusion product used to assess this model of ZnT-1 orientation in the membrane showed that the C terminus of ZnT-1 was indeed intracellular. The rat ZnT-1 gene was mapped to chromosome 1.

ZnT-1 has one consensus site for N-linked glycosylation that would be facing extracellularly, as well as consensus sequences for other types of posttranslational processing, including sites for protein kinase C phosphorylation and a site for phosphorylation by a tyrosine kinase. Helical wheel projection of each of the putative membrane spanning regions suggests some amphipathic nature to regions 1, 2, and especially 5, which could perhaps form a core or channel through which zinc could be translocated.

Expression of ZnT-1 with either a c-myc epitope or as a green fluorescent fusion protein localized ZnT-1 to the plasma membrane. Moreover, cells overexpressing ZnT-1 were much more resistant to high concentrations of extracellular zinc and exhibited lower intracellular zinc levels (as measured by a metal-responsive reporter construct). These observations led to the hypothesis that ZnT-1 functions as a zinc exporter. Cells extremely sensitive to extracellular zinc had the lowest zinc efflux capacity, whereas cells overexpressing ZnT-1 had a relatively high zinc efflux capacity, and the parental cell line had an intermediate efflux capacity, which supports that hypothesis (Palmiter and Findley 1995). Zinc regulation of ZnT-1 expression may occur by a metal response element (MRE) in the promoter.

Dependence of efflux conferred by ZnT-1 on factors such as ionic gradients, energy, and cognate metals has been examined. Zinc efflux capacity related to ZnT-1 did not require a sodium gradient, suggesting that it may not be a sodium-dependent transport process. Similarly, zinc efflux was not sensitive to azide. This suggests that ATP is not required; therefore ZnT-1 is not likely to act as or be coupled to an ATP dependent pump. ZnT-1 had a low affinity for the transport of other metals, such as cadmium and copper, demonstrating specificity for zinc (Palmiter and Findley 1995). Taken together, these data suggest that the overexpression of ZnT-1 confers on cells the ability to efflux zinc in a nonsodium- and nonenergy-dependent manner and that the action of this protein may be at the level of the plasma membrane (Fig. 1). However, it should be noted that, until the expression of ZnT-1 can be demonstrated to influence zinc transport in a heterologous expression system such as the Xenopus oocyte, it would be premature to definitively define ZnT-1 as a species responsible for zinc translocation across the lipid bilayer. Alternatively, ZnT-1 may be an auxiliary factor that stimulates transport or may be a subunit of a larger zinc transporter complex (Palmiter and Findley 1995).

View larger version (164K): [in this window] [in a new window]   Fig 1. Tissue specificity and cellular locations for putative mammalian zinc transporters. Intracellular free Zn(II) concentration, probably < 1 nmol/L, is shown in light blue. Intracellular Zn potentially compartmentalized in vesicles is shown in olive. ZnT-1 is located at the plasma membrane and functions as an exporter for zinc efflux in virtually all organs. ZnT-2 may be associated with vesicular zinc uptake or cellular export in many organs. ZnT-3 is associated with vesicular zinc uptake in neurons and testis. ZnT-4 probably is located at the plasma membrane of mammary gland and brain. DCT1 is a metal transporter with an affinity for iron, zinc, and other cations that functions as a cellular importer for metal uptake by many organs. Localized by immunochemistry to basolateral membrane of enterocytes (*) and renal tubular cells (**) or to synaptic vesicles (***).

Tsuda et al. (1997) have examined the regulation of ZnT-1 by transient forebrain ischemia and its potential role in delayed neuronal death. In cloning genes regulated by forebrain ischemia by differential display PCR, a partial sequence of the gerbil homologue of ZnT-1 was isolated. This led to the cloning of the full-length ZnT-1 cDNA. The gerbil and rat ZnT-1 sequences exhibit high homology (92%). A brief ischemic period resulted in a marked increase in ZnT-1 mRNA in the CA1 pyramidal neurons. The level of zinc in this neuronal area also was elevated during this same time period, suggesting that zinc might be either responsible for or the result of the upregulation of ZnT-1 mRNA expression. When primary neuronal cultures were generated and exposed to high levels of extracellular zinc, ZnT-1 mRNA levels also were elevated, indicating that the activating pathway had been preserved in this cell culture model.

Regulation of zinc efflux by cells of various organs is one mechanism by which zinc levels are retained during periods of diminished dietary zinc supply and that enhances zinc release when the supply is elevated. Consequently, ZnT-1 expression should be influenced by changes in zinc intake. McMahon and Cousins (1998) demonstrated that intestinal ZnT-1 mRNA is increased when rats are fed a high zinc level (180 mg Zn/kg diet) compared with the normal zinc level (30 mg Zn/kg) found in the American Institute of Nutrition 76A/93 diet formulations (Reeves et al. 1993). A deficient zinc intake (5 mg Zn/kg) did not reduce ZnT-1 expression. Affinity purified antibodies against a carboxy terminal peptide of ZnT-1 suggest that ZnT-1 protein from intestine and liver migrates as a 42- and 36-kDa protein, respectively. These antibodies showed that changes in ZnT-1 protein from membrane preparations in response to elevated dietary zinc intake were similar to those also found for ZnT-1 mRNA. A high acute dose of zinc given by gavage markedly increased ZnT-1 expression. However, the levels of ZnT-1 mRNA and protein do not always coincide, which suggests ZnT-1 may be a component of a more complex efflux system or have a secondary role in efflux, perhaps at very high intakes. Davis et al. (1998) showed that in mice that overexpress (transgenic) or do not express (knockout) the metallothionein gene, the expression of intestinal and hepatic ZnT-1 was similar, suggesting that expression is not dependent upon the level of this major zinc-binding protein. However, in both genotypes, ZnT-1 mRNA levels are influenced markedly by acute zinc administration in a fashion similar to metallothionein mRNA. This further supports the concept that the ZnT-1 gene is regulated by an MRE in the promoter.

In the small intestine, ZnT-1 is highly expressed in the duodenum and jejunum and not in the ileum or colon (McMahon and Cousins 1998). Furthermore, as shown in Figure 2A, ZnT-1 is localized at the basolateral surface of rat enterocytes lining the villi and is not found in goblet cells or those cells within the lamina propria. This location and regional distribution is consistent with a function for ZnT-1 in zinc acquisition and/or retention. Furthermore, because ZnT-1 expression is not decreased by low zinc intake, it is likely that this transporter does not have a role in an organism's ability to adapt to such dietary conditions to maintain cellular zinc concentrations. ZnT-1 also is located at the basolateral membrane of renal tubular cells (Fig. 2B). This places ZnT-1 in an orientation that would be expected of a transporter that functions in zinc reabsorption by the renal tubules. It has been well established that urinary zinc output is minimal under normal physiologic conditions (Cousins 1996, King and Keen 1994) because of extensive tubular reabsorption (Victery et al. 1981). As ZnT-1 expression and localization in other tissues is examined, its potential contribution to overall zinc homeostasis at the integrative level will be further clarified.

View larger version (143K): [in this window] [in a new window]   Fig 2. Localization of ZnT-1 transporter at basolateral membrane of enterocytes from villi of rat small intestine (A, longitudinal section) and renal tubular cells (B, cross-section). Affinity purified rabbit IgG against a peptide from the N-terminal end of rat ZnT-1 was the primary antibody. A fluorescently tagged secondary antibody was used for localization with an epifluorescence microscope. Magnifications are ×1000. lp, lamina propria; g, goblet cell; n, nucleus; l, lumen. Arrowheads designate basolateral membrane.

ZNT-2 Transporter.  ZnT-2 was identified by the same selection method used to isolate the ZnT-1 sequence (Palmiter et al. 1996a). ZnT-2 is predicted to be composed of 359 amino acids and shares only 26% homology with ZnT-1. The length of ZnT-2, however, has not been definitively determined because of the absence of a traditional start codon. Despite the difference in length, ZnT-2 also is predicted to contain six membrane-spanning regions and also a shortened histidine-rich region, possibly as an intracellular loop. The ZnT-2 gene has been mapped to rat chromosome 4, demonstrating that the ZnT gene family is not arranged in genetic proximity (Fig. 1).

Transfection of the ZnT-2 gene into cells only slightly lowered activity of a zinc-responsive reporter gene construct. This implied that cytosolic zinc was not being eliminated extracellularly. However, transfection allowed the cells to survive in much higher concentrations of extracellular zinc, suggesting that the extra zinc was diverted to a compartment that precluded cell damage. Localization of a ZnT-2/green fluorescent fusion protein by microscopy indicated a punctate appearance, suggesting intracellular targeting or compartmentalization. In addition, cells transfected with ZnT-2 and grown in high concentrations of extracellular zinc exhibited a highly vacuolated appearance. These vesicles were acidic, as measured by the uptake of pH-sensitive dyes, and exhibited simultaneous localization of zinc (as measured by the zinc-specific fluorescent chelator Zinquin) (Palmiter et al. 1996a). Acidified vesicles were not required for the uptake of zinc into these compartments, as demonstrated by the persistence of zinc with raised vesicular pH. This suggests that a proton gradient is not required for uptake of vesicular zinc. The cell system used for these experiments lacked expression of both metallothionein and ZnT-1. When these two genes were expressed along with ZnT-2, the appearance of the vesicles was markedly attenuated. Thus the definitive physiological role of ZnT-2 awaits definition.

ZNT-3 Transporter.  Library screening yielded the third zinc transporter gene, ZnT-3 (Palmiter et al. 1996b). ZnT-3 exhibits more homology to ZnT-2 (49%) than ZnT-1 (18%) and is predicted to contain six transmembrane-spanning regions. The gene for ZnT-3 has been mapped to mouse chromosome 5. Expression of ZnT-3 in a zinc-sensitive cell type did not result in a more zinc-resistant phenotype in contrast to the phenotypic alterations induced by ZnT-1 and ZnT-2 expression. The expression of ZnT-3 is restricted to the brain and testis, implying some specific role in these two tissues (Fig. 1). ZnT-3 expression in the brain appears to be limited to the synapses and axons of glutamatergic neurons, suggesting an important role for zinc at these sites. In agreement with this proposal is the colocalization of ZnT-3 and reactive zinc in the macromolecular brain region known as mossy fiber boutons (Wenzel et al. 1997). ZnT-3 was shown to be targeted to vesicles in the synaptic termini of glutamatergic neurons, implicating a role in zinc packaging for triggered release from synaptic vesicles (Palmiter et al. 1996b). Zinc was postulated to have an influence on neuronal cell death during ischemia (Koh et al. 1996). Consequently, changes in ZnT-3 could alter that process and other zinc-dependent factors associated with neurodegenerative diseases. Vesicular packaging of zinc also could be important in spermatogenesis and insulin secretion (da Silva and Williams 1991). Both could involve ZnT-3 transporter activity associated with vesicles.

ZNT-4 Transporter.  In a search for the mutant mouse gene pallid, using positional cloning, Huang and Gitscher (1997) isolated the sequence for ZnT-4 located on chromosome 2. ZnT-4 shares the greatest homology with ZnT-2 and ZnT-3, 67 and 62%, respectively. It also exhibits characteristics of the zinc transporter family; six predicted transmembrane regions, a histidine-rich region thought to be oriented intracellularly, and both the amino and carboxy termini residing intracellularly. Expression of ZnT-4 in a yeast cell line that was zinc-sensitive restored zinc resistance (Huang and Gitscher 1997), implicating this protein is involved in zinc efflux or compartmentalization (Fig. 1). A single point mutation resulting in a premature termination is now known to cause lethal mouse (lm) syndrome. This mutation is characterized by the inability of pups nursed by lm mothers to survive to weaning (Piletz and Ganschow 1978). Before succumbing, pups display features of zinc deficiency, including alopecia and stunted growth. This can be prevented by zinc administration to the pups or the lm mother or, alternatively, nursing the pups on foster dams (Ackland and Mercer 1992). Decreased zinc transport from the mammary gland of lm dams to their milk was identified as the cause of this lethal genotype (Lee et al. 1992). Consequently, identification of the ZnT-4 gene as the defect in the lm genotype implicates this transporter in the deposition of zinc into the maternal milk supply. However, ZnT-4 expression in other tissues, including a high level of expression in the brain, suggests a more far reaching role in zinc metabolism.

DCT1 Transporter.  Because metal transporters have not as yet been well characterized at the biochemical level, it is not known whether these transporters are, in all cases, monospecific. Instead, some may exhibit transport capability for a spectrum of metals. It is for that characteristic we have included DCT1 in this review. Gunshin et al. (1997) used a classical approach toward transporter identification to clone and characterize DCT1. Size-fractioned poly(A)+ RNA from rat duodenum was injected into Xenopus oocytes and ⁵⁵Fe uptake was measured. Subsequently, a specific cDNA was isolated from a cDNA library that produced ⁵⁵Fe uptake activity. DCT1 is a 561 amino acid polypeptide with 12 transmembrane-spanning regions and glycosylation sites in the fourth extracellular loop. An intracellular loop is similar to a motif involved in ATP-coupling in bacterial transport systems (Fig. 1).

In duodenum, DCT1 mRNA levels are highest in enterocytes lining the crypts and lower villi rather than villus tips. DCT1 expression is increased markedly by nutritional iron deficiency. Oocytes showed a change in membrane current, indicative of transport, that was correlated with DCT1 expression. Zn, Cd, Mn, and Cu had substrate specificities slightly greater than Fe in producing current changes. DCT1 has high homology to a family of mammalian genes, the macrophage Nramp family. The latter are involved in host defense mechanisms through unknown biochemical functions.

Collectively, the tissue specificity and metal transporter activity support a role for DCT1 in zinc transport. DCT1 expression may increase in iron deficiency to aid in iron acquisition. DCT1 is the rat homologue of human Nramp 2. The latter has been proposed as being essential for both intestinal iron absorption and iron uptake by erythroid cells (Fleming et al. 1998). The well-recognized nutritional interaction between zinc and iron (Cousins 1996, King and Keen 1994) could be explained in part on the basis of a multi-ion function for this transporter.

	SUMMARY AND PERSPECTIVES

The description of a family of transporters helps bridge the integrative and reductionist approach to the study of zinc transport. Although four members have now been described, it would not be surprising if more members of this protein family emerge. Additionally, other transporters that may exhibit a capacity to transport several metal congeners, such as DCT1, may contribute to both the observed total activity of zinc transport as well as to mineral-mineral interactions. Most of the studies, except the experiments regarding ZnT-1 and ZnT-3, have been performed in either isolated cell culture or in heterologous cell systems. Evidence for a role of any ZnT protein in the actual transport of metals across the lipid bilayer is wanting. Expression of these proteins in heterologous expression systems like the Xenopus oocyte will provide strong evidence that these proteins are the transporting molecules themselves. Clearly, a careful examination of the role and contribution of each of these transporters to overall zinc absorption and homeostasis in the intact animal also should be viewed as a priority. Furthermore, the response of these genes to other factors, including immune challenge, oxidative damage, or varying levels of other metals that compete for these transporters also should be considered. Collectively, a careful description of characteristics of transporters that influence the uptake as well as efflux of zinc and factors that influence their regulation will provide a clearer understanding of the molecular events that regulate zinc homeostasis.

	ACKNOWLEDGMENTS

The authors thank Virginia Mauldin and Walter Jones for the artwork.

	FOOTNOTES

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