Keywords

At the time a most comprehensive review was published on “the biochemical basis of zinc physiology,” the basis was the then known structures and functions of zinc proteins (Vallee and Falchuk 1993). A quarter of a century later, we can record a heightened impact of zinc on biology and significant advances that include a further impressive increase in the number of zinc proteins, roles of zinc ions in signal transduction, and major insights into how cellular zinc is controlled homeostatically (Rink 2011). How zinc is regulated cannot be discussed without considering how the other essential metal ions are regulated. Each metal ion is controlled in a certain range of affinities so that it can function specifically without interference with the others. Zinc is the second-most competitive divalent metal ion next to copper, which is tightly controlled by cellular metallochaperones. Critical for the biological actions of zinc is its high affinity to binding sites in proteins, leaving very little zinc unbound. The affinities are in the picomolar or even femtomolar range (Maret 2004a; Kluska et al. 2018). Based on simple mass equilibria, the resulting steady-state “free” zinc ion concentrations are expected to be picomolar as indeed confirmed experimentally (Krężel and Maret 2006). These low concentrations are not negligible. They are functionally important and tightly controlled. Total cellular zinc concentrations are remarkably high. They are hundreds of micromolar, putting them in the range of those metabolites such as ATP. Therefore, zinc is not a trace element in the cell. In this chapter, I shall describe how biological control of zinc is achieved, why it is so complex, and how zinc regulates a multitude of cellular processes at several hierarchical levels.

Zinc biology is based on the zinc ion (Zn2+), which is redox-inert in biology, and its interaction with proteins. Hence, we need to be concerned only with one valence state, which is often referred to as “zinc” although the word stands for the element (Zno). Despite not participating directly in the redox biology of living organisms, zinc affects redox metabolism and redox signaling indirectly (Maret 2019). Zinc coordination occurs with the nitrogen, oxygen, and sulfur donors of the side chains of mainly four amino acids: His, Glu, Asp, and Cys, and only occasionally others. Coordination is flexible as zinc does not underlie the geometric constraints governing the coordination of the transition metal ions. Therefore, a variation of the number and the types of coordinating donors can modulate the chemical characteristics so that zinc can serve many different functions. A remarkable property is that the sulfur donor of cysteine confers redox activity on zinc coordination environments. It makes this redox-inert metal ion part of redox metabolism (Maret and Vallee 1998). Oxidoreduction of the sulfur allows biological control of protein functions through reversible zinc binding and mobilization of zinc from its tight binding sites (Maret 2006). It is not the only way of conferring mobility on zinc, though. While zinc is bound permanently during the lifetime of many proteins, it binds transiently to some proteins that have sites with coordination dynamics to enhance dissociation rates and move zinc ions (Maret and Li 2009; Maret 2011a). Thus, coordination environments also have a role in biological time and determine whether zinc resides in the protein for a very long time, for example, carbonic anhydrase with a half-life of about half a year, or is moved relatively fast during transfer to other proteins or transport through membrane proteins (Maret 2012). For example, transport rates of zinc transporters are on a 10–100 ms time scale, many orders of magnitude faster than the zinc dissociation rates of zinc from zinc metalloproteins (Chao and Fu 2004). This mobility is achieved by protein dynamics and reducing or not having scaffolding of sites, a term that refers to fastening donor ligands in the primary coordination sphere through interactions with a secondary coordination sphere. The coordination environments of the zinc ion when not bound to proteins are not known. Because of the abundance of low molecular weight molecules that could potentially serve as ligands, the coordination chemistry in the cell is much more complex than simple aqueous solution chemistry (Krężel and Maret 2016). The roles of non-protein ligands in the control of cellular zinc are not known either, but glutathione is a major candidate (Marszałek et al. 2018).

Zinc has functions in cellular signaling pathways that include proliferation and differentiation of cells (Beyersmann and Haase 2001). At the molecular level, its functions are based on catalytic, structural, and regulatory roles in proteins. It includes binding sites between proteins or their subunits (Maret 2004b). The first two functions are well characterized in numerous zinc metalloproteins. Owing to its ability to organize protein domains, zinc contributes significantly to the diversity of protein architecture and biomolecular interactions. Although zinc regulation has been mentioned in the literature for a long time, the details and the extent of regulatory functions of zinc were unknown, mainly because there was no knowledge about how such regulation with a metal ion that is tightly bound to proteins is possible. The sheer number of zinc proteins is impressive and makes zinc the most important metal ion for protein structure and function. Bioinformatics approaches defined the zinc proteome (Andreini et al. 2006). Humans have an estimated 3000 zinc proteins. This count does not include sites with regulatory functions, the number of which is unknown as will be discussed later (Maret 2008).

All of these cellular functions of zinc require an efficient regulatory system. A quantitative concept of how multicellular eukarya regulate cellular zinc and how zinc can regulate protein functions is the subject of two recent articles. In the previous edition of this book, the molecular aspects of how cellular zinc and zinc ion transients are handled were discussed (Maret 2014). The cellular aspects involve a high degree of compartmentalization, intracellular trafficking, and different types of zinc ion signals (Maret 2017). The present article builds on the content of these articles and provides an update on the cellular and molecular mechanisms of how zinc is regulated and how it can serve as a signaling ion.

2.1 Proteins Regulating Zinc: Buffering and Muffling in Cellular Zinc Homeostasis

Key to understanding the control of zinc is the affinity of proteins for zinc and the resulting available “free” zinc ion concentrations. The control of “free” metal ion concentrations is commonly described by metal ion buffering in analogy to proton buffering. The pKa of acid-base pairs and the ratio of their concentrations determine the pH value. Likewise, the pKd of ligands for zinc and the ratio of bound and unbound ligands determine the pZn value. With the relatively high affinities of cellular zinc-binding sites in zinc proteins, the resulting “free” zinc ion concentrations are very low, yielding pZn values of ten or higher (Krężel and Maret 2006). Another parameter that is important for control is the zinc buffering capacity. It describes how resistant the pZn is to change. It is in the micromolar range in cells (Krężel and Maret 2006).

In biology, in contrast to simple solution chemistry, removal or addition of zinc by transport processes also contributes to metal buffering of a cell. This property is referred to as muffling (Colvin et al. 2010). Minimally, two dozen zinc transporters, a dozen metallothioneins, and at least one zinc sensor, metal-regulatory element (MRE) binding transcription factor-1 (MTF-1), participate in cellular zinc homeostasis. This remarkably large number of proteins is needed to regulate zinc ion transients (fluctuations) in addition to ascertaining proper zinc concentrations and to distribute zinc subcellularly for its functions in organelles. An important aspect of the subcellular biology of zinc is its storage in vesicles (zincosomes), and the role of zinc-containing vesicles in cellular exocytosis of zinc ions for extracellular functions.

2.2 Metallothioneins

Metallothionein (MT) has been known for 70 years (Margoshes and Vallee 1957). The protein, thionein (T), obtained its name from being sulfur-rich. It binds metal ions, hence the name metallothionein for the metal-bound form. Its function was thought to be elusive for a long time. In fact, it could not have been defined because knowledge about how cellular zinc is regulated and what exactly the affinities of MT for zinc are did not exist. Zinc coordination in MT is entirely based on interactions with the sulfur donors of cysteines in 2 “clusters,” 1 binding 3 zinc ions to 9 cysteines and the other 4 zinc ions to 11 cysteines (Robbins et al. 1991). Inorganic biochemists pondered long over what purpose such a binding in a unique structure might have in biology. The answer came from determining the affinities of MT for its seven zinc ions and relating them to the affinities of other proteins for zinc as well as to the “free” zinc ion concentrations. The answer is that MT is a regulated biological zinc (and copper) buffer (Krężel and Maret 2017).

MT has not just one high affinity for zinc as previously thought and which would make it a storage molecule. Instead, its affinities for zinc vary over a wider range, exactly the range needed for buffering cellular zinc (Krężel and Maret 2007). The dissociation constants (10−11 and 10−9 M) are commensurate with the measured “free” zinc ion concentrations of a few hundred picomolar. The affinities of some sites of MT for zinc are weaker than those of zinc for zinc-requiring metalloproteins, thus leaving the large number of zinc proteins requiring zinc for function in the zinc-bound state in the presence of MT. MT can be compared to a “clean sweeper,” keeping zinc away from spurious, weaker binding sites, but at the same time ascertaining that genuine zinc metalloproteins obtain and retain their zinc. On the other hand, the induction of T, changing the MT/T (bound/unbound ligand) ratio, removes zinc from proteins that have weaker zinc-binding sites than genuine zinc metalloproteins and can bind zinc when zinc ion concentrations increase (Krężel and Maret 2008). The fact that the binding sites of MT are not saturated with zinc under normal physiological conditions, as expressed by an MT/T ratio that varies as a function of zinc availability, supports a function as a zinc buffer (Krężel and Maret 2008).

Zinc buffering of MT is regulated. On the protein level it is redox regulation (Maret 2011b). The thiols in thionein are coupled to redox systems (Sagher et al. 2006; Maret 2006). In this way, redox reactions can control the zinc chelating capacity of MT, making more zinc available under oxidizing conditions when “Tox” is formed and making less zinc available under reducing conditions when “Tred” is formed (Fig. 2.1) (Maret 2000). Regulation also occurs on the gene level. In humans, about a dozen genes code for MTs and they are differentially regulated (Li and Maret 2008). A host of signaling pathways, in particular stress signals, control the total concentration of MT and indicate the large number of processes associated with managing cellular zinc. A significant number of transcription factors controlling MT gene expression are zinc proteins (Krężel and Maret 2017).

Fig. 2.1
figure 1

Zinc buffering. At least a dozen human genes code for thioneins that bind zinc ions – and other ions such as cuprous ions – in their reduced form (Tred) and form metallothioneins (MT). MT is usually not fully saturated with zinc ions as required for a buffer, a property described with an MT/T ratio. The protein is coupled to redox reactions. Oxidation forming partially oxidized thionin (Tox) reduces the zinc-binding capacity of the protein and makes zinc available, while reduction of thionin forming Tred reduces zinc availability. Many different pathways regulate the expression of the thionein genes, in particular many chemical and physical stressors, and include a large number of zinc-containing transcription factors (ZnTFs). One feedforward regulation is the induction of thionein at increased zinc ion concentrations via the MTF-1 transcription factor. It generates zinc chelating and reducing capacity. Further regulation is afforded via methylation of the promoters of thionins

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One could posit that other proteins and ligands with zinc-binding capacity buffer zinc as well. They certainly do but tighter or weaker binding is not relevant for the physiological range of buffering zinc for signaling, just as, for example, only one of the three pKa values of phosphoric acid is relevant for pH buffering in a particular range. MT does not work in isolation. It is a “rheostat” for zinc, linking cellular signal transduction pathways to MTF-1 sensing and gene expression (Hardyman et al. 2016). Only if zinc buffering in MT is exhausted, MTF-1 is activated for making more thionein, exporting zinc ions, and for other purposes.

MT is also involved in the cellular translocation of zinc. During the cell cycle, it moves from the cytosol to the nucleus (Nagel and Vallee 1995). It also translocates from the cytosol to the mitochondrial inter membrane space (Ye et al. 2001).

In addition to this dynamic zinc buffering by MT (Fig. 2.1), muffling involves transport of zinc out of the cell or into a cellular compartment (Fig. 2.2). This process is also rather complex with multiple importers and exporters and their regulation. It is required for compartmentalization and control of zinc ion signals in addition to overall homeostatic control of cellular zinc.

Fig. 2.2
figure 2

Co-operation of zinc transporters. Plasma membrane transporters of the Zip family import extracellular zinc, [Zn2+]e, while transporters on intracellular membranes of the ZnT family distribute intracellular zinc, [Zn2+]i, subcellularly. In this directional presentation of zinc flux, the use of the two types of transporters in the export pathway is opposite to the one in the import pathway. Both import from outside the cell and import from cellular compartments increase “free” zinc ion concentrations via Zip transporters while ZnT transporters decrease them

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2.3 Zinc Transporters

In contrast to MT, the zinc coordination chemistry in the transport sites of two families of zinc transporters, Zips and ZnTs, does not involve sulfur donors. 3D structures of the mammalian proteins are unknown and all inferences about their functions are made from the 3D structures of bacterial homologues. While all of them transport zinc, their specificity is poorly characterized as some family members can transport other metal ions, in particular iron and manganese.

The impressive number of 24 zinc transporters illustrates how important it is to regulate cellular zinc (Fukada and Kambe 2011; Kambe et al. 2015). Zinc transporters need to regulate zinc ion transients in addition to maintaining the correct levels of zinc: some Zips control the formation of zinc ion signals; ZnTs restore the steady-state (Fig. 2.3). Most of the Zip transporters are located on the plasma membrane though some can localize intracellularly. With the exception of ZnT1 and the evolutionary related ZnT10, ZnTs localize to intracellular membranes. Different cellular compartments require specific acquisition and delivery of zinc and transport against various concentration gradients. To accomplish this, zinc transporters may use different ions such as bicarbonate, calcium, or protons in symport or antiport (Gaither and Eide 2000; Girijashanker et al. 2008; Levy et al. 2019). The transporters are secondary active transporters; none of them are ATP-dependent. At least for one ZnT, it has been shown that it can couple to a proton gradient generating pump. The high concentration of zinc in exocytotic vesicles requires an active process as indeed shown for ZnT2 being coupled to the vacuolar ATPase (Lee et al. 2017). Tissue-specific expression of zinc transporters and removal from the plasma membrane and degradation affords further regulation.

Fig. 2.3
figure 3

Zinc muffling. In addition to the regulation of zinc ions via the metallothionein/thionein buffering system (Fig. 2.1), two families of zinc transporters participate in regulating cellular zinc by import, distribution, and export, and in providing zinc-dependent proteins with zinc. The transporters have different activities, such as being an androgen receptor (Zip9), loading enzymes of the secretory pathway with zinc (ZnT5,6), or providing exocytotic vesicles with zinc, [Zn2+]v, (ZnT2,3,8). In addition, zinc release from the endoplasmic reticulum (ER) via Zip7 can induce intracellular signaling leading to enzyme inhibition. Extracellular zinc signaling to another cell is achieved through exocytosis and binding to a zinc receptor (ZnR, GPR39), which stimulates intracellular calcium [Ca2+]i release

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2.3.1 ZnTs (Solute Carrier Family SLC30A1-10)

ZnTs belong to the cation diffusion facilitator (CDF) family of proteins. 3D structures have been reported for the E. coli protein YiiP and a related protein from Shewanella oneidensis (Lu and Fu 2007; Lopez-Redondo et al. 2018). These bacterial proteins have additional metal-binding sites believed to sense zinc ions for export. For mammalian proteins, zinc sensing mechanisms are unknown, however.

The bacterial proteins are dimers with six α-helices in the transmembrane domain (TMD) in the monomer and with a more variable, relatively large C-terminal cytoplasmic domain (CTD) with an αββαβ structure, except for ZnT9. The CTD has the capacity to bind several metal ions and to serve as a hub for interactions with other proteins. Whether zinc binding to the CTD is permanent for maintaining its structure or transient for either delivering zinc ions to the TMD or sensing them is not known. Bacterial CDFs work by conformational changes with an alternative access mechanism and proton antiport and serve as a model for mammalian ZnTs, but significant differences exist (Lu et al. 2009). While the bacterial proteins export an excess of zinc ions from cells, eukaryotic cells require zinc to be re-distributed intracellularly when there is no known excess of zinc ions. How the intracellular transporters acquire zinc from the low steady-state picomolar “free” zinc ion concentrations is unknown. The majority of ZnTs are within organellar membranes and serve diverse functions, such as filling vesicular stores and supplying organelles and secreted proteins with zinc and loading exocytotic vesicles with zinc for fundamental biological processes, among which neurotransmission (ZnT3), insulin storage (ZnT8) , and lactation (ZnT2) are the best characterized.

Overall, the core structure of the TMD and CTD is relatively well conserved but significant differences exist in the different members regarding extensions at the N- and C-termini and additional loops between TM helices. ZnT5 has twice the number of TM helices. The phylogenetic tree shows clades consisting of the vesicular transporters (ZnT2-4, ZnT8), ZnT1 and ZnT10, ZnT5 and ZnT7, and ZnT6 and ZnT9 (Hogstrand and Fu 2014). ZnT6 does not transport zinc but heterodimerizes with ZnT5 when supplying zinc for zinc proteins of the secretory pathway (Tsuji et al. 2017).

Characterization of the individual CTD of human ZnT8 shows that it folds independently and that its metal binding is different from that of the bacterial transporters, binding only two instead of the four or six metal ions at the dimer interface in the bacterial transporters (Parsons et al. 2018).

2.3.2 ZIPs (Zrt/Irt-Like Proteins) (Solute Carrier Family SLC39A1-14)

A 3D structure has been determined for a ZIP transporter from Bordetella bronchiseptica. The TM helices have a novel 3+2+3 arrangement (Zhang et al. 2017). The zinc transport site is a binuclear metal site. Human Zip2, however, is predicted to have a mononuclear metal transport site and shows pH and voltage modulation of metal transport (Gyimesi et al. 2019). The 3D structure of the extracellular domain of human Zip4 was determined in an approach to understanding the entire mammalian protein by piecing together the structures of individual domains (Zhang et al. 2016). It forms two subdomains with a linker, which is characteristic of all nine LIV1 members with the so-called PAL motif. The extracellular domain forms a dimer. Some Zips form heteromers (Taylor et al. 2016). Two other parts of the Zip4 molecule were investigated regarding zinc binding, and they are believed to participate in zinc sensing, an extracellular loop with histidines (Chun et al. 2019) and an intracellular loop with histidines (Bafaro et al. 2019). The intracellular loop is thought to sense zinc ion concentrations that are sufficiently high and signal no further need for zinc import. This sensing is linked to removal of Zip4 from the plasma membrane and degradation (Shakenabat et al. 2015).

The phylogenetic tree encompasses one major clade, the LIV1 subfamily, containing 12/4; 8/14; 5 & 10/6; 13/7 and another clade containing Zip1-3. Zip11 and Zip9 are more distantly related. Zip9 is an androgen receptor and zinc transporter (Thomas et al. 2018).

2.4 MTF-1

MTF-1, metal regulatory element (MRE)-binding transcription factor-1, controls zinc-dependent gene expression. The protein has six zinc fingers which are implicated in sensing increased zinc ion concentrations (Laity and Andrews 2007). It responds to stressors such as heavy metals, hypoxia, and oxidative stress and induces the expression of thionein and ZnT1 to maintain cellular zinc homeostasis. It is essential for embryonic liver development (Günther et al. 2012). Another emerging aspect of MTF-1 is that it regulates miRNAs via MREs and thus another level of regulation of zinc metabolism (Francis and Grider 2019).

2.5 Signaling with Inorganic Ions: Ca2+ and Zn2+

Phosphate metabolism is critical for energy metabolism. Because the strong interaction of phosphate with calcium can cause precipitation of calcium phosphate, free calcium concentrations must be kept very low in the cell compared to the environment. Zinc ions, because of their generally higher affinity for ligands, are kept at even lower concentrations, but they must be acquired from an environment low in zinc. It has been stated that signaling substances occur at rather low concentrations for energetic reasons (Carafoli and Krebs 2016). For both zinc and calcium, it is achieved through buffering by proteins and exquisite regulation. Uncontrolled increases are a major pathway for cytotoxicity and cell death. “Free” zinc ion concentrations are about three orders of magnitude lower than the already low steady state free calcium concentrations, although the total concentrations of the two ions differ at the most only by one order of magnitude: calcium a few millimolar, zinc hundreds of micromolar. This difference allows the two signaling systems to co-exist and complement each other (Fig. 2.4). Measuring the lower concentrations of “free” zinc with sufficiently selective probes and sensors has been experimentally challenging. The biological functions of the two metal ions have a lot in common (Table 2.1). And the two signaling systems co-operate. Zinc ions inhibit the Ca2+-ATPase on the plasma membrane with a Ki value of 80 pM and thus exert control over cellular-free calcium concentrations (Hogstrand et al. 1999). Another target of zinc is the cardiac ryanodine receptor/calcium channel (RyR2), which leaks calcium when cellular “free” zinc ion concentrations increase above 2 nM (Reilly-O’Donnell et al. 2017). Together with Mg2+, the three redox-inert metal ions can regulate processes over many orders of magnitude in concentrations. Its redox inertness and flexible coordination environments make zinc ideally suited for a role as a messenger and for transmitting specific signals to sites on proteins.

Fig. 2.4
figure 4

Signaling with redox-inert metal ions. Zinc signaling complements calcium signaling but occurs in a separate, much lower range of concentrations. Both signaling systems interact with redox and phosphorylation signaling

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Table 2.1 Ca2+ vs Zn2+ as carriers of signals
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2.6 Zinc Regulating Proteins (Zinc/Zn2+ Signaling)

2.6.1 Intracellular Regulation

How can the very low concentrations of available “free” zinc ions regulate and serve as signaling ions in a zinc-buffered environment? When fluctuations of cellular zinc ion concentrations were measured, it became apparent that zinc ions indeed regulate biological functions (Fukada et al. 2011). Specific mechanisms exist for signal generation, transduction, and decoding. Two pathways are known to elicit cellular zinc ion transients. One is the release of zinc ions from an intracellular store (Yamasaki et al. 2007; Taylor et al. 2012a) and the other is the release of zinc from zinc/thiolate sites in proteins such as in metallothioneins. Release of zinc from a store in the ER occurs in response to the hormone stimulation of cells, which results in casein kinase-2-dependent phosphorylation and opening of the Zip7 channel (Taylor et al. 2012b). The released zinc is required for skin (dermis) and B-cell development through controlling ER zinc, and hence ER stress, and cytosolic zinc (Bin et al. 2017; Woodruff et al. 2018; Anzilotti et al. 2019) (Fig. 2.3). Casein kinase-2 is a zinc protein with zinc finger-mediated dimerization of the regulatory subunit (Chantalat et al. 1998). Whether other Zip transporters are regulated in a similar way is not known. Stimulation also occurs with zinc ions, indicating that there is a zinc-induced zinc release just as in the case of calcium (Kjellerup et al. 2018). Release of zinc from zinc/thiolate coordination environments occurs under conditions of redox signaling and oxidative or carbonyl stress (Hao and Maret 2006). It can lead to Zn2+-dependent gene expression, which is part of a feedback loop as it includes proteins involved in chelating zinc and in an antioxidant response. Aside from zinc-dependent gene expression, other molecular targets of the zinc ions have not been linked directly to the events that cause a zinc transient. In contrast to activation of gene expression, the typical response is an inhibition of enzymes (Maret et al. 1999). The overall effect can be confusing because zinc inhibition on the molecular level can result in the activation of a process. For example, a general observation is that an increase of cellular free zinc ions enhances phosphorylation signaling (Haase and Maret 2003). One cause for this activation is an inhibition of protein tyrosine phosphatases (PTPs), which are the main regulators of phosphorylation signaling. In PTP1B, the phosphatase controlling the insulin and leptin receptors, zinc inhibits only the enzyme in the phosphorylated form through the generation of a zinc-binding site in the closed conformation (Bellomo et al. 2014, 2016). The inhibition constant for receptor protein tyrosine phosphatase β (RPTPβ) is 21 pM, suggesting that some PTPs are inhibited tonically and need to be activated by zinc removal (Wilson et al. 2012). Many other enzymes with inhibition constants in the nanomolar range have been reported (Maret 2013a). One common feature is that catalytic residues positioned in proximity can bind a metal ion, that is, cysteine-dependent enzymes with a nearby histidine. Such diads and triads of amino acids in the active site have dual functions: They serve as catalytic groups and they provide the donors for zinc binding and inhibition. Inhibitory zinc-binding sites on several enzymes, including caspases, cathepsins, and kallikreins, have been characterized structurally (Maret 2013a; Eron et al. 2018). The inhibition involves specific mechanisms of binding and regulation such as allosterism and binding one or several zinc ions. The inhibition of many proteinases indicates a role of zinc in proteostasis. Since these zinc-inhibited enzymes do not need zinc for activity they are not considered to be zinc enzymes although their zinc binding is almost as strong as that for genuine zinc enzymes. Because of the strong metal inhibition, the enzymes are often supplied commercially with the chelating agent ethylenediaminetetraacetic acid (EDTA) to keep them active, thus masking the zinc inhibition. While most zinc sites in zinc proteins are readily recognized by signatures/motifs with characteristic sets of donors linked by relatively short non-coordinating amino acid spacers, this is not the case in inhibitory sites. Therefore, these sites are not amenable to the database mining approach that has been so successful in predicting the total number of zinc metalloproteins in the human genome (Andreini et al. 2006). Consequently, we do not have estimates of the number of regulatory sites. An even less understood facet is how the zinc-inhibited proteins are activated. At least two ways can be envisaged. One is lowering the free zinc ion concentrations by muffling, removing zinc ions with transporters, and the other is buffering, binding zinc by a protein such as thionein. In the first case, the re-activation would depend on the rate of zinc dissociation, which could be limiting unless assisted by coordination dynamics of the proteins as in the zinc transport sites of zinc transporters. In the second case, zinc dissociation would be fast if a chelating agent accelerates it but slow if gene expression such as induction of thionein is involved. To distinguish the two possibilities, one would need to know the duration of the cellular zinc ion transients in addition to their amplitudes, which are in the order of a few nanomolar when measured globally. The link between zinc ion signals and their targets is often tenuous and it remains unknown whether local zinc ion transients or a global change of the pZn value is responsible for the effect. Linking zinc ion transients with specific targets is one of the pressing issues in the field of zinc signaling to explain the great number of functional readouts that have been reported.

An exception to the rule that the action of each metal is well separated from that of others is the binding of zinc to magnesium sites. Work dating back to 1961 showed that zinc inhibits Mg-dependent phosphoglucomutase with an inhibition constant (IC50) of about 2 pM (Milstein 1961). The tight inhibition was then used to set an upper limit to the “free” zinc ion concentration possible without inhibiting the enzyme (Peck and Ray 1971). Investigations into whether zinc is indeed a physiological modulator of the activity of this enzyme have not been pursued. Also, Mg2+ activates PTP1B while Zn2+ inhibits it (Bellomo et al. 2018). Another relationship between the two essential minerals is that some kinases prefer a Zn-ATP complex over the typically employed Mg-ATP complex (McCormick 2002).

2.6.2 Extracellular Regulation

In addition to intracellular signaling, there is extracellular zinc ion signaling. It was first described in neurotransmission and established zinc ions stored in synaptic vesicles (boutons) as a neuromodulator (Frederickson et al. 2005). The best described target is the N-methyl-D-aspartate (NMDA) receptor. Its structure shows zinc in the high affinity (5 nM) binding site in the N2A subunit bound to His-44, His-128, Glu-266, and Asp-282. Only two ligands bind zinc at the low affinity site (7.5 μM): His-127 and Glu-284 (Romero-Hernandez et al. 2016).

In addition to the zinc-glutamatergic neurons, many other cells have the capacity to secrete zinc ions. They do so for different purposes with paracrine, autocrine, or even endocrine effects. One target is ZnR/GPR39, a G-protein coupled receptor on the plasma membrane activating phospholipase β, releasing Ca2+ from the ER, and stimulating extracellular signal-regulated kinases (ERK1/2) and protein kinase B (Akt) (Hershfinkel 2018). Fertilized mammalian embryos emit zinc sparks. The exocytotically released zinc ions are coordinated with cellular calcium transients and modify the structure of the zona pellucida to avoid polyspermy (Que et al. 2019). The ZnT2,3 and 8 zinc transporters are involved in loading exocytotic vesicles with zinc. The exocytosed zinc ions in other tissues and their targets have received comparatively less attention. Zinc affinities for extracellular proteins probably do not need to be as low as for those in the cytosol. Zinc binds to and affects the function of a large number of membrane channels, but only few of them have affinities in the nanomolar range (Peralta and Huidobru-Toro 2016). The conditions for buffering zinc ions are different in the extracellular environment, the vesicles, and the cytosol. Therefore, intracellular sites on membrane proteins may need higher affinities for zinc modulation than extracellular sites.

2.7 Zinc Functions in Health and Disease

Zinc is involved in virtually all aspects of cellular functions and is important for the control of proliferation, differentiation, and programmed cell death (Maret 2013b). On an organismal level, it is essential for growth, development, maintenance of function, and protection against various physical and chemical insults. The implications of perturbation of zinc metabolism for disease are huge. The role of zinc in diseases can originate from mutations, the number of which is quite high in zinc transporters and MTs regulating cellular zinc (Hogstrand and Maret 2016; Raudenska et al. 2014). Several diseases are specifically caused by a disruption of zinc transporter function and thus perturbation of zinc homeostasis: acrodermatitis enteropathica (Zip4), neonatal transient zinc deficiency (ZnT2), and SCD-Ehlers-Danlos Syndrome (Zip13). In many more diseases or syndromes, zinc is involved causally. Nutritional zinc deficiency and interference of xenobiotics with zinc metabolism are also causes of diseases. Discovery of the numerous molecular functions of zinc has been a boon for biochemistry but a bane for a wider acceptance of the overall importance of zinc for the cell biology. Reasons for the latter are the lack of a biomarker for the cellular functions of zinc and the overall issue that pleiotropic functions of zinc may give the impression of a lack of specificity thus concealing its true importance.