Zn²⁺ at a cellular crossroads

Abstract

Zinc is an essential micronutrient for cellular homeostasis. Initially proposed to only contribute to cellular viability through structural roles and non-redox catalysis, advances in quantifying changes in nM and pM quantities of Zn²⁺ have elucidated increasing functions as an important signaling molecule. This includes Zn²⁺-mediated regulation of transcription factors and subsequent protein expression, storage and release of intracellular compartments of zinc quanta into the extracellular space which modulates plasma membrane protein function, as well as intracellular signaling pathways which contribute to the immune response. This review highlights some recent advances in our understanding of zinc signaling.

Introduction

Zinc is essential for cellular homeostasis. More specifically, ten percent of all genes code for zinc-binding motifs [1]. Zinc dyshomeostasis can lead to cell death. Once believed to be essential solely for structural and catalytic functions in proteins and enzymes, zinc recently has been recognized as a signaling agent. Specific stimuli are hypothesized to trigger a transient increase in free zinc (Zn²⁺), which subsequently interacts with target molecules in a signal transduction process. In contrast to alkali and alkali earth metal-mediated signaling, the term free zinc does not necessarily denote hydrated metal ions, which adds complexity to these events. Instead Zn²⁺ exists in a weakly bound form that is more accessible than protein-bound zinc [2]. Transient Zn²⁺ increases can be generated by efflux from vesicles (zincosomes) or changes in cellular redox potential mediated by cytosolic proteins [3]. Metal ion buffering and muffling are the two critical parameters that determine zinc availability and therefore the nature of signaling process [4].

Estimates place the eukaryotic resting free Zn²⁺ concentrations in the picomolar range depending on the cell type [5-7], although quantifying these exceedingly low concentrations remains a significant technical challenge [8,9]. Since the total concentrations of zinc in cells is between 200-300 μM [10], maintaining homeostasis requires complex cellular machinery. This includes transcription factors which are turned on or off by small changes in the cellular concentration of Zn²⁺ as well as proteins that are marked for degradation through the ubiquitination pathway when cellular zinc levels increase for example [11,12]. Both thermodynamic and kinetic models of homeostasis are necessary to understand zinc maintenance. In the thermodynamic model pZn describes the physiological zinc potential [5]. Analogous to pH, the value of pZn is determined by the K_d of the buffering ligand(s) (pZn = −log[Zn²⁺]). Strongly buffered zinc implies that high affinity ligands maintain low free Zn²⁺ concentrations. Under these conditions, a transient increase in Zn²⁺ concentration will return to resting concentrations quickly. When buffering capacity is weak, cells reach a new steady-state Zn²⁺ concentration. To produce lasting Zn²⁺ signals, buffering capacity must be lower since signals would be terminated quickly at high pZn.

Muffling denotes the time-dependent redistribution of internalized Zn²⁺ under non-steady state conditions, and the subsequent return to resting cytosolic concentrations [10,13]. Removing surplus zinc from the cytoplasm by muffling enables cells to increase zinc uptake, despite a lack of buffering capacity. The temporal component of muffling process, as well as the ability for cells to temporarily tolerate non-steady state Zn²⁺concentrations are important factors in signaling.

Cellular Zinc Entry

Two classes of zinc transporters control the concentration of zinc in the cytosol as well as in intracellular compartments. Zinc Transport (ZnT) proteins function to decrease the cytosolic concentrations, while Zinc- or Iron-regulated transport Proteins (ZIP) increase the cytosolic concentrations [14]. While ZIP transporters are members of the cation diffusion facilitator class of proteins, ZnT proteins are not. Comparison of the crystal structure of YiiP, a bacterial ZnT protein with an ab initio model of the human (h) ZIP4 transporter suggests that both protein families have a similar transmembrane metal coordination site [15,16]. Both ZnT and ZIP proteins are expressed on the plasma membrane as well as on the membranes of intracellular organelles. Therefore, both ZnT and ZIP proteins control zinc trafficking between cellular compartments and zinc availability. One of these proposed intracellular compartments, the zincosomes, are loaded with Zn²⁺, usually by ZnT3. Furthermore, zincosomes may interact with receptors on cell membranes [17,18].

In addition to zinc-specific routes of entry mediated by ZnT and ZIP transport proteins, several ion channels may conduct Zn²⁺ under specific conditions. Under normal resting conditions blood plasma has sub-nM free Zn²⁺ [19]. However, transient increases in extracellular free Zn²⁺ have been observed, most commonly in the synaptic cleft. Under these conditions, some ion channels can use the electrochemical gradient to conduct Zn²⁺. For example, voltage-gated calcium channels control Ca²⁺ cell entry in response to membrane potential changes [20]. More specifically, the dihydropyridine-sensitive L-type calcium channel (LTCC) contributes to Zn²⁺ import in cortical neurons [21], β-cells in pancreas islet [22], and Zn²⁺ release from the endoplasmic reticulum in mast cells [23]. Ionotropic glutamate receptors (GluRs) are ligand-gated ion channels expressed mainly in the central nervous system (CNS) that mediate most excitatory synaptic transmission in mammalian brains [24]. Various GluR subunits may provide Zn²⁺ entry routes in postsynaptic neurons and astrocytes during neuronal excitation [7,25,26]. In addition, both acetylcholine receptors (AChR) [27] and transient receptor potential (TRP) channels have been shown to conduct Zn²⁺ [25].

Zinc-Mediated Modulation of Membrane Protein Function

Glutamate-gated NMDA receptors play a crucial role in neuronal communication, synaptic plasticity and memory formation [28,29]. The NMDA receptor is a dimer (GluN1A and GluN2B) where each subunit consists of an amino-terminal domain (ATD), a ligand-binding domain (LBD) and transmembrane domains (TMD) [30]. Upon the requisite stimulus, zinc quanta can be released from zincosomes within presynaptic terminals into the synaptic cleft (Figure 1). The exposed ATD can bind allosterically active ligands including Zn²⁺ found in the extracellular space. The inhibitory role of Zn²⁺ on NMDA receptors was recognized when micromolar Zn²⁺ in cultured hippocampal neurons reduced the amplitude of excitatory postsynaptic potentials by 50% compared to controls [31]. Subsequent analysis revealed that Zn²⁺ inhibits NMDA directly by two different mechanisms. High-affinity Zn²⁺ binding to the ATD of GluN2A subunit stabilized closed-cleft conformations, which reduced channel open probability. Low-affinity binding to the TMD is voltage-dependent and transiently blocks current flow through open channel [32].

Figure 1.

A representation of extracellular Zn²⁺ signaling pathways. Zn²⁺ released into the extracellular space between cells can interact with proteins on the surface of another cell. Zn²⁺-binding can initiate a variety of responses including the release of Ca²⁺, another signaling metal ion. For example, signaling between neurons occurs when Zn²⁺ is released from vesicles in the presynaptic neuron into the synapse. Interactions between Zn²⁺ and protein receptors on the postsynaptic neuron are the next step in the signaling cascade.

The G-protein-coupled zinc sensing receptor (ZnR/GPR39) is expressed in the pancreas, gastrointestinal tract, liver, adipose tissue and the central nervous system. ZnR signaling contributes to cell proliferation, differentiation and/or death. [33-35] Unlike the inhibitory effect on the NMDA receptors upon zinc coordination, ZnR/GPR39 is an activator. Zn²⁺ coordination is mediated through a tridentate Zn²⁺-binding site on the extracellular domain which is comprised of two histidine and one aspartic acid residue [36]. Zn²⁺ coordination to ZnR triggers Ca²⁺ release via the IP3 signaling pathway [37].

Protein-Mediated Intracellular Zn²⁺ Release

In contrast to extracellular Zn²⁺ signaling which is initiated upon zinc release from an intracellular compartment into the extracellular space, intracellular Zn²⁺ signaling can be triggered by metal ion release from cytosolic proteins. A transient increase in intracellular Zn²⁺ is dependent upon oxidation of protein ligands since the metal ion is redox-inactive under physiological conditions. Consequently, a redox signal is converted into a Zn²⁺ signal. As an example, metallothioneins (MTs) bind up to seven Zn²⁺ in two thiolate-rich sites. The α-domain (C-terminal), which binds four metal ions, has a higher affinity for Zn²⁺, while the β-domain (N-terminal) coordinates up to three more labile metal ions [38]. At least four different MTs have been identified including MT-3 that is widely distributed in the CNS [39-41]. Nitric oxide, which has also been identified as an important signaling molecule, can oxidize the cysteine thiolate ligands, releasing a zinc signal [42].

Metal-responsive transcription factor-1 (MTF-1) is a zinc-finger transcription protein which regulates metal-responsive gene expression [43,44]. MTF-1 is the main activator for MT expression as well as select ZnTs [45,46]. MTF-1 contains six Cys₂His₂ zinc fingers (ZFs) which recognize and bind to DNA metal responsive elements (MRE) [47,48]. Zn²⁺, which can be acquired directly from mobile pools in the cytosol or indirectly by Zn²⁺ release from MTs by either cadmium or oxidative stress, activates MTF-1 upon coordination to ZFs [49,50]. Activated MTF-1 then localizes to the nucleus, binds to the MRE and alters protein expression levels on the transcriptional level [51].

Representative Signaling Events Trigger by Intracellular Zn²⁺ Release

Extracellular stimuli such as hormones, growth factors and cytokines can trigger zinc transients in the cytosol [18]. Transient changes in the intracellular concentration of Zn²⁺ initiate second messenger pathways through early (fast) zinc signaling (EZS) or late zinc signaling (LZS, Figure 2i) [52]. EZS is transcription-independent where the timescale ranges from seconds to minutes. In contrast, LZS requires the transcription of zinc transport proteins with longer lasting effects (hours, Figure 2ii).

Figure 2.

A representation of various intracellular Zn²⁺ signals. In fast Zn²⁺ signa ling (i), binding of an extracellular ligand can trigger the release of Zn²⁺ from intracellular stores, liberation of Zn²⁺ from MTs by oxidative processes or flow of Zn²⁺ through permeable channels. Interaction of Zn²⁺ with target receptors that occurs before homeostasis is restored, constitutes a signaling pathway. Alternatively, in late Zn²⁺ signaling (ii), import and export by proteins changes the intracellular concentrations, which triggers transcriptional regulation of the homeostasis machinery.

Toll-like receptors (TLRs) contribute to the innate immune response by recognizing microbes [53]. TLRs are widely expressed on the surface of immune cells such as dendritic cells and macrophages, as well as on non-immune cells such as fibroblasts and epithelial cells [54-56]. When TLR-4 is activated it oligomerizes and recruits Toll/IL-1 receptor domain-containing adaptors to initiate signal transduction pathways that culminate in the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ΚB) and mitogen-activated protein kinases (MAPKs) [57-60]. NF-ΚB is a transcription factor which contributes to multiple physiological and pathological conditions, including the apoptosis, carcinogenesis, immune response and inflammation. MAPKs function to phosphorylate serine, threonine and tyrosine. Phosphorylation mediated by MAPKs contributes to initiating the immune response.

Zn²⁺ produces a dual effect on TLR-4 induced cytokine secretion [61]. Stimulation of human leukocytes with lipopolysaccharide (LPS) results in a moderate cytosolic Zn²⁺ increase. Trapping Zn²⁺ with N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) suppresses MAPK stimulation. Zn²⁺-induced MAPK activation occurs within 2 min [62]. LPS can also decrease labile Zn²⁺, and high Zn²⁺ concentrations inhibit TLR-4 signals [63-66]. Zn²⁺ may be involved in TLR-4 signaling, regulating myeloid differentiation primary response gene 88 via EZS, and TIR-domain-containing adapter-inducing interferon-β signaling by modulating basal Zn²⁺ concentrations [67]; however, there is no direct evidence Zn²⁺ activates MAPK. While the kinase might not be the molecular target for Zn²⁺, Zn²⁺ inhibits MAPK phosphatases (MKPs) [68], so the specifics of the putative Zn²⁺ signaling mechanism have yet to be elucidated.

Fcε receptor I (FcεRI) belongs to the family of multi-chain immune recognition receptors (MIRRs) [69], and is found in mast cells, basophils and eosinophils [70,71]. Upon extracellular stimulus of FcεRI by immunoglobulin E, a rapid increase in intracellular Zn²⁺, or zinc wave is observed [72]. This zinc wave regulates downstream signaling pathways and enhances the transcription of cytokines and affects several processes, including tyrosine phosphatase activity [18].

Zinc Sparks

Cell cycle arrest at metaphase II (MII) of meiosis is a key step in mammalian oogenesis. Upon fertilization, a series of Ca²⁺ oscillations activate downstream molecular targets [73,74]. The impact of Zn²⁺ on the eukaryotic cell cycle was first recognized three decades ago [75,76], but recently, O'Halloran reported Zn²⁺ uptake by mouse oocytes during maturation [77]. After the onset of Ca²⁺ oscillations, Zn²⁺-enriched oocytes eject Zn²⁺ into the extracellular milieu in a series of coordinated zinc sparks. The process lasts for hours and facilitates cell cycle resumption. Quantitative maps of the fluxes reveal that zinc sparks originate from vesicles. These vesicles undergo dynamic movement during oocyte maturation and exocytosis upon fertilization [78]. Two zinc transport proteins, ZIP6 and ZIP10, are expressed in the cortex and have been proposed to be responsible for Zn²⁺ accumulation [79]. Cumulus cells also may contribute to the zinc transport network by forming cumulus-oocyte complex [80].

Zinc sparks mediate egg-to embryo transitions [65,77]. Equally, Zn²⁺ may exert a concentration-dependent regulation of meiosis through an interaction with the zinc-binding EM12, a central component of the cytostatic factor (CSF) [81]. EM12 contains a C-terminal zinc-binding region that inhibits the anaphase promoting complex/cyclosome [82-84]. When intracellular Zn²⁺ reaches a threshold concentration, coordination to the zinc binding region activates APC/C inhibitor EM12, which leads to CSF-mediated MII arrest. After activation, zinc sparks reduce both cellular zinc and EM12 activity, which drives egg activation [81].

Conclusions

Zn²⁺ signaling is mediated by transient changes in free zinc. Extracellular Zn²⁺ signaling can take place in extracellular environment such as the synaptic cleft where Zn²⁺ released from the presynaptic cleft modulates protein function on postsynaptic neurons. Intracellular Zn²⁺ signaling has been observed in many cell types and is generated in the cytosol from the extracellular space or from intracellular Zn²⁺ stores. Cytosolic proteins such as metallothioneins may also contribute to the Zn²⁺ signals through redox chemistry. The import, export and trafficking of Zn²⁺ by proteins all contribute to a cells capacity to generated, utilize and terminate signals. The ongoing efforts to study these various processes are vital to mapping signal transduction pathways mediated by Zn²⁺.

Highlights to be used.

Zinc transport proteins decrease cytosolic concentrations

Iron-regulated transport proteins increase cytosolic zinc concentrations

Extracellular zinc release is involved in a variety of signaling processes

Intracellular zinc release can be involved in signaling by binding to receptors or regulate protein at the transcriptional level

Zinc sparks can control egg to embryo transitions