Mechanism and Regulation of Cellular Zinc Transport
- 41 Downloads
Zinc is an essential cofactor for the activity and folding of up to ten percent of mammalian proteins and can modulate the function of many others. Because of the pleiotropic effects of zinc on every aspect of cell physiology, deficits of cellular zinc content, resulting from zinc deficiency or excessive rise in its cellular concentration, can have catastrophic consequences and are linked to major patho-physiologies including diabetes and stroke. Thus, the concentration of cellular zinc requires establishment of discrete, active cellular gradients. The cellular distribution of zinc into organelles is precisely managed to provide the zinc concentration required by each cell compartment. The complexity of zinc homeostasis is reflected by the surprisingly large variety and number of zinc homeostatic proteins found in virtually every cell compartment. Given their ubiquity and importance, it is surprising that many aspects of the function, regulation, and crosstalk by which zinc transporters operate are poorly understood. In this mini-review, we will focus on the mechanisms and players required for generating physiologically appropriate zinc gradients across the plasma membrane and vesicular compartments. We will also highlight some of the unsolved issues regarding their role in cellular zinc homeostasis.
Active Zinc Transport Across the Cell Membrane
Ion gradients are generated by two main mechanisms: 1) A primary pump, utilizing the energy of ATP-hydrolysis; or 2) a secondary active mechanism that uses an ion gradient, such as Na+, for generating Zn2+ gradients. A Zn2+ pump has been demonstrated in bacteria, where several forms of p-type ATPases have been shown to catalyze active Zn2+ transport (24). Recently, a similar ATPase, which transports Zn2+ and Cd2+ and to a lesser extent other heavy metals, has been discovered in Arabidopsis (25,26). Surprisingly, there is still no evidence for a Zn2+ pump in either yeast or mammalian cells, though a Cu pump has been identified that is linked to heavy metal ion transport (27).
A Na+-dependent secondary active mechanism has, however, been suggested to facilitate formation of the transmembrane Zn2+ gradient in neurons. Early studies suggested that the neuronal Na+/Ca2+ exchanger mediates Zn2+ extrusion (28), but more recent findings seem to support the existence of a distinct Na+/Zn2+ exchanger. These studies have indicated that a putative Na+/Zn2+ exchanger, probably a member of the Na+/Ca2+ exchanger superfamily, operates with a stoichiometry of 3Na+/1Zn2+, promoting Zn2+ efflux against a 500-fold transmembrane gradient (29). This mechanism is pharmacologically and molecularly distinct from the classical Na+/Ca2+ exchangers. Whether this exchanger is the principle plasma membrane extruder of Zn2+ or is accompanied by an as yet unidentified Zn2+ pump, is an open and intriguing question.
The Role of ZnT Proteins in Cellular Zinc Homeostasis
ZnT proteins- expression and physiological roles.
Altered expression phenotype
Overexpression: reduced [Zn2+] i and enhanced resistance against Zn2+ toxicity, KO: Lethal at embryonic stage,
small intestine, kidney, placenta, pancreas, testis, seminal vesicles, and mammary gland
Overexpression: enhanced lysosomal and vesicular Zn2+ accumulation
Synaptic vesicles (Glutamatergic and GABAergic)
KO: synaptic Zn2+ deficiency, enhanced susceptibility to seizure, loss of gender specific Alzheimer’s disease plaque formation in a mouse model, decreased susceptibility to amyloid angiopathy
mammary gland, brain, small intestine and mast cells
Lethal milk syndrome in mice, Asthma (mice), Alzheimer’s disease
Pancreatic β-cells, intestine, heart brain, liver, kidney
Insulin secretory vesicles, Golgi Spliced isoform: plasma membrane Complexed with ZnT-6
KO: poor growth, osteopenia, male specific fatal arrhythmias. Essential for folding and secretion of Zn2+ -binding enzymes.
liver, brain, and small Intestine
Complexed with ZnT-5
Alzheimer’s disease (mice)
small intestine, liver, retina, spleen, kidney, and lung
Essential for folding and secretion of Zn2+ -binding enzymes,
Insulin secretory vesicles
Polymorphism marker in diabetes type II
Overexpression: enhances glucose dependent insulin secretion
Although the ZnTs are increasingly recognized as critical players in cellular zinc homeostasis, for the sake of brevity, only a few examples will be discussed.
ZnT-1, a ubiquitously expressed member of the SLC30 zinc transporter family, is found on the plasma membrane of neurons (37) and glia cells (38,39). In the mouse brain, ZnT-1 is, in general, localized in regions rich in synaptic Zn2+ (though cerebellar Purkinje cells are also intensely ZnT-1-immunoreactive) and its expression is developmentally regulated in correlation with the appearance of the synaptic Zn2+ (39,40). ZnT-1 has been shown to reduce Zn2+ toxicity in neurons and glial cells (37,38,41). Expression of this protein is highly regulated by Zn2+, via the transcription factor MTF-1 (32), and priming of glial cells with non-toxic Zn2+ exposures promotes ZnT-1 expression (38). Thus, sublethal exposure to Zn2+ might induce ZnT-1 (and perhaps MT) expression to counteract subsequent (toxic) rises in [Zn2+]i. However, it must be emphasized that the mechanism by which ZnT-1 maintains low [Zn2+]i is more complex than previously thought.
Possibly the most extensively-studied ZnT, ZnT-3 is localized to the membranes of Zn2+-containing vesicles in glutamatergic synaptic boutons (44). Support for the idea that this transporter is essential for uploading Zn2+ into synaptic vesicles comes from ZnT-3 knockout mice, which lack chelatable Zn2+ throughout the brain. While early studies on this mutant failed to discern a distinct phenotype (45,46), later studies, focusing on brain disorders, such as stroke, epileptic seizure, and Alzheimer’s disease, have demonstrated a role for synaptic Zn2+, e.g., increasing susceptibility to seizures (47, 48, 49). An intriguing question emerging from this work is whether synaptic Zn2+ participates directly in fundamental processes of synaptic transmission such as LTP. Two recent studies (50,51) show that LTP in the cortico-amygdala pathway and hippocampus, respectively, does require Zn2+ release, while others (e.g.16,52) have indicated that LTP is not required. Although this issue is still unresolved, the ZnT-3 KO model continues to provide an excellent tool to examine these questions.
ZnT-5 and ZnT-6
ZnT-5 and ZnT-6, vesicular ZnTs found on Golgi and ER, are thought to functionally interact (53,54). Deletion of the ZnT-5 gene leads to abnormal bone development, weight loss, and lethal, male-specific, cardiac arrhythmias (55). Interestingly, one of the hallmarks of zinc deficiency is cardiac dysfunction (56). Several studies have indicated that ectopically expressed ZnT-5 or ZnT-6 are capable of independently transporting Zn2+, while others suggest that the activity of the Zn2+-dependent protein, TNAP (tissue-nonspecific alkaline phosphatase), requires their hetero-oligomerization (53, 54, 55,57,58). While this links ZnT5/6 and vesicular Zn2+, the precise mechanism involved and their regulation remain poorly understood. Studies on yeast and bacterial ZnT homologues, considered together with the distribution of mammalian ZnTs in acidic cellular compartments, suggest that intracellular ZnTs are linked to both, Zn2 and H+ transport. Results from our group indicate that ZnT-5 is capable of independently mediating Zn2+ transport, but coexpression of ZnT-5 and ZnT-6 accelerates its rate. We have further demonstrated that Zn2+ transport mediated by ZnT-5 is linked to changes in Golgi pH (Ohana et al. in preparation).
Localization of splice variants of ZnT-5 at various stations in cells (59) suggests a more versatile role in intracellular zinc homeostasis then was previously envisioned.
ZnT-8 is exclusively expressed in pancreatic β-cells (60). Zinc is essential for the proper processing and packaging of insulin into secretory vesicles. Indeed, recent studies indicate that heterologous expression of ZnT-8 results in enhanced insulin accumulation and secretion (61). Furthermore, it has been shown recently that a polymorphism of ZnT-8 in humans is linked genetically to susceptibility to Type II diabetes (62). It should be noted that other ZnT family members, among them ZnT-5 and ZnT-6, are also found on secretory vesicles, influencing insulin production and secretion. Thus, the question is whether a functional interaction occurs between these Zn2+ transporters, and what their specific role in insulin production and secretion is.
Additional Questions and Challenges for Future Research
As seen by the brief discussion above, the characterization of multiple ZnT proteins has not yet led to elucidation of a mechanism of action for most of them. Many fundamental questions remain about their activity and regulation, as well as their interaction with other ion transporters. Of particular interest is the question of how these proteins are capable of transporting zinc against the vanishingly low intracellular concentration existing in mammalian cells. One possible mechanism is direct buffering of Zn2+ by the ZnTs. Alternatively, the transporters may interact with metallothioneins (MT) which will transfer zinc to the ZnTs. A similar mechanism has been shown for the shuttling of Fe3+ and Cu2+ (63). Finally, release of zinc from metallothioneins, in response to specific intracellular signals (e.g., NO) or ischemia, may also provide free Zn2+ to the ZnTs.
Another question regards the existence of functional crosstalk between the Zn2+ transporters and other ion transport mechanisms, particularly proteins involved in maintaining cellular pH homeostasis, such as the Na+/H+ exchangers. This is especially relevant considering the putative mechanism of H+/Zn2+ exchange, which may underlie the activity of the intracellular ZnTs.
The structural organization of the ZnTs also is poorly understood at this time. Hetero-oligomerization, has been suggested to underlie the activity of at least some of the transporters. Is this phenomenon exclusive to ZnT5 and ZnT6, or is it shared by other ZnT members? Such interactions would increase significantly the functional repertoire of zinc transport proteins. The functional interaction between ZnT-3 and the vesicular glutamate transporter, Vglut1, would support such a mechanism (64).
Finally, at the dawn of the era of proteomics and cellular networking, is it possible to begin drawing the network map of the zinc transport proteins? Powerful tools to address these issues, e.g., fluorescent zinc sensors targeted to specific organelles, have been developed and are being constantly refined (65, 66, 67, 68, 69). The advantage of such an approach has been demonstrated previously for H+ and Ca2+ using GFP-based chameleon and pHluorin constructs. Based on seminal studies performed on yeast and mammalian cells, the feasibility of studying zinc in organelles using such an approach is promising, (65,70). The results will have an impact far beyond cellular zinc homeostasis.
Thanks to the many members in the zinc community for their invaluable insight and discussions. This work was supported in part by Binational Science Foundation Grant 2001101 (to M. H.), Israel Science Foundation Grant 456/02.1 (to I. S.), German Israel Foundation (GIF, project nr. I-588-99.1/1998 to I.S.), and Israel Science Foundation equipment Grant 456/02.2 (to I. S.).
- 63.Prohaska JR, Gybina AA. (2004) Intracellular copper transport in mammals. JNutr. 134:1003–6.Google Scholar