**4. Correlations between calcium and heavy metal exposure as seen in** *S. cerevisiae* **cells**

When grown in media contaminated with heavy metals, the yeast cell wall is the first to get in contact with the surplus cations present in the cell surroundings. If the contamination is not excessive, the cations would probably get stuck at this level, due to the mannoproteins that compose the outer layer of the cell wall (alongside of β-glucans and chitin) which are heavily phosphorylated and carboxylated, decorating the cell façade with a negatively charged shield prone to bind to positively charged species, such as the metal cations [83]. Excess metal ions which escape the negatively charged groups on the cell wall surface penetrate the porous cell wall and reach the membrane to exert their toxic effect by disrupting the lipid bilayer or by assaulting the membrane transporters.

Several heavy metals (Co2+, Cu2+, Fe2+, Mn2+, Ni2+, and Zn2+) are essential for life in their ionic forms, acting mainly as cofactors for a variety of enzymes. They are necessary only in minute amounts inside the cell (hence their denomination as "trace" elements); if their concentration goes beyond the physiological threshold they become toxic by nonspecifically binding to any biomolecule bearing a negative charge or a metal-chelator fragment. The bipolar nature of trace metals determined the development of intricate cellular systems dedicated to their uptake, buffering, sequestration, intracellular trafficking, compartmentalization and excretion. As in many other directions of study, *S. cerevisiae* brought a considerable contribution to the understanding of the molecular mechanism involved in trace metal transport and homeostasis [3, 38–47]. Several heavy metal transporters were identified at the plasma membrane level (**Figure 4A**), with both high and low affinity. For example, Ctr1p, Smf1p and Zrt1p are involved in the high-affinity uptake of Cu<sup>+</sup> , Mn2+ and Zn2+, respectively [84–86]. Low-affinity plasma membrane transporters are more numerous and less specific: Fet4p for Fe2+, but also for Cu2+, Cd2+, Mn2+, and Zn2+; Zrt2p for Zn2+, but also for Fe2+, Co2+, Cu2+, Cd2+, Mn2+ [87, 88]. Transporters for phosphate or amino acids were also shown to participate in the low-affinity transport of Cd2+, Co2+, Cu2+, Mn2+, and Ni2+ [89, 90]. All these transporters are likely to be assaulted by surplus metals (**Figure 4B**) when cells are exposed to contaminated environments [91].

To have any chance of survival under heavy metal stress, the cell needs to be one step ahead of the "villain" ions and to get prepared for defense by using various strategies. The attempts

cytosol with the exception of La3+ (lanthanum) and to a lesser extent, Pr3+ and Nd3+ [81]. Care must be taken when using Ln3+ as channel blockers, as it was shown that at low concentrations

**Figure 3.** Transgenic aequorin as a tool for measuring intracellular Ca2+. A. Schematic representation of aequorin bioluminescence [72–74]. Cells expressing apo-aequorin are first incubated with the cell-permeant coelenterazine to produce functional aequorin. When Ca2+ binds to aequorin, the protein undergoes a conformational change leading to the destabilization of the peroxide group (-O-O-), linking apoaequorin to coelenterazine, decomposing it to to coelenteramide

; the coelenteramide, which is in an excited state, generates blue light (λmax = 469 nm). B. Schematic representation of Ca2+-induced bioluminescence of yeast cells expressing reconstituted aequorin in the cytosol. When cells are exposed to an insult (e. g., environmental stress) the secondary messenger Ca2+ ions enter the cytosol and bind to aequorin, rendering

cyt wave [75, 76].

When grown in media contaminated with heavy metals, the yeast cell wall is the first to get in contact with the surplus cations present in the cell surroundings. If the contamination is not excessive, the cations would probably get stuck at this level, due to the mannoproteins that compose the outer layer of the cell wall (alongside of β-glucans and chitin) which are heavily phosphorylated and carboxylated, decorating the cell façade with a negatively charged shield prone to bind to positively charged species, such as the metal cations [83]. Excess metal ions which escape the negatively charged groups on the cell wall surface penetrate the porous cell

**4. Correlations between calcium and heavy metal exposure as seen** 

Ln3+ may leak into the cytosol via the Cch1p/Mid1p system [82].

the cell luminescent. Luminescence traces indicate the intensity and the duration of the [Ca2+]

**in** *S. cerevisiae* **cells**

and CO<sup>2</sup>

28 Calcium and Signal Transduction

**Figure 4.** Toxicity of heavy metal exposure. A. Schematic representation of transporters involved in the uptake of essential metals under normal conditions. B. Under high surplus of heavy metals, the transporters will carry the excess cations into the cell, where they bind non-specifically to biomolecules, altering their structure and functionality [91].

to understand the role of calcium in preparing the yeast cell to resist the heavy metal attack are summarized in the following sections.

determined that divalent Cd2+ and Ca2+ have very similar physical properties, with ionic radii of Ca2+ (0.97 Å) and Cd2+ (0.99 Å) giving similar charge/radius ratios, meaning that these ions are able to exert strong electrostatic forces on biological macromolecules [95]. Under such circumstances, the Cd2+-induced aequorin luminescence observed could also be the result of aequorin binding to Cd2+ instead of Ca2+. This was not the case though: when measuring the Cd2+ accumulation in yeast cells, it was revealed that the Cd2+-induced aequorin luminescence occurred significantly faster than the Cd2+ uptake, indicating that the luminescence produced

Cu2+ is one of the most important essential metals: a variety of enzymes require copper as a cofactor for electron transfer reactions [96]. Nevertheless, when in excess, Cu2+ is very toxic in the free form because of its ability to produce free radicals when cycling between oxidized

could be alleviated by Ca2+ [97]. The role of Ca2+ in mediating the cell response to high concentrations of Cu2+ was investigated in parallel with Cd2+, and it was noted that exposure to high Cu2+ determined broad and prolonged [Ca2+]cyt waves which showed a different pattern from the [Ca2+]cyt pulses induced by high Cd2+ [23]. In contrast to Cd2+, Ca2+ − mediated responses to

It was found that the cell exposure to high Cu2+-induced broad Ca2+ waves into the cytosol which were accompanied by elevations in cytosolic Ca2+ with patterns that were influenced by the Cu2+ concentration but also by the oxidative state of the cell [18, 24]. When Ca2+ channel deletion mutants were used, it was revealed that the main contributor to the cytosolic Ca2+ pool under Cu2+ stress was the vacuolar Ca2+ channel, Yvc1p, also activated by the Cch1pmediated Ca2+ influx (**Figure 6**). Using yeast mutants defective in the Cu2+ transport across the plasma membrane, it was found that the Cu2+-dependent Ca2+ elevation could correlate with the accumulated metal, but also with the Cu2+ − induced oxidative stress and the overall

Ca2+-mediated responses to external stress [24]. Interestingly, other redox active metals such as Mn2+ or Fe2+ were inactive in inducing [Ca2+]cyt waves ([23], unpublished observations),

High manganese failed to elicit Ca2+ elevations irrespective of the magnitude of the insult applied ([23]; unpublished observations). The response was monitored over a wide range of concentrations (from the quasi-physiological 0.5 mM to the super lethal 50 mM) and times (up to 60 min of exposure). Of all the cations, Mn2+ is the closest to Ca2+ in terms of ionic radius and charge. This similarity is so relevant that Mn2+ effectively supports yeast cell-cycle progression in place of Ca2+ [99]. This similarity probably renders the cell irresponsive to high concentrations of an otherwise toxic metal. A more subtle Mn2+-Ca2+ interplay exists though, being

been known that the inhibitory effect of Cu2+ on glucose-dependent H<sup>+</sup>

high Cu2+ depend predominantly on internal Ca2+ stores [24] (**Figure 6A**).

oxidative status. Moreover, it was revealed that Cu2+ and H<sup>2</sup>

probably because these metals are less redox-reactive than the Cu2+/Cu<sup>+</sup>

. Studies correlating Ca2+ with Cu2+ toxicity in yeast are scarce, but it had

Calcium and Cell Response to Heavy Metals: Can Yeast Provide an Answer?

http://dx.doi.org/10.5772/intechopen.78941

31

O2

efflux from *S. cerevisiae*

acted in synergy to induce

couple (**Figure 6D**)

was the result of increase in [Ca2+]cyt [23].

**4.2. Cu2+**

Cu2+ and reduced Cu<sup>+</sup>

under aerobic conditions [98].

**4.3. Mn2+**

### **4.1. Cd2+**

Cd2+ is one of the most studied non-essential heavy metals as it is a global environmental pollutant present in soil, air, water, and food, representing a major hazard to human health [92]. External Cd2+ was shown to unequivocally induce the [Ca2+] cyt elevations in *S. cerevisiae*, as recorded in aequorin-expressing cells, which responded through a sharp increase in the [Ca2+]cyt, just a few seconds after being exposed to high Cd2+ [23]. Interestingly, the chemically similar Zn2+ and Hg2+ failed to elicit [Ca2+]cyt elevations under the same conditions [23]. The response to high Cd2+ depended mainly on external Ca2+ (transported through the Cch1p/ Mid1p channel) and to a lesser extent on the vacuolar Ca2+ (released into the cytosol through the Yvc1p channel). The adaptation to high Cd2+ was influenced by perturbations in Ca2+ homeostasis in that the tolerance to Cd2+ often correlated with sharp Cd2+-induced [Ca2+]cyt pulses (**Figure 5A**, **B**), while the Cd2+ sensitivity was accompanied by the incapacity to rapidly restore the low levels of [Ca2+]cyt [23] (**Figure 5C**).

It had been suggested that Cd2+ toxicity was a direct consequence of Cd2+ accumulation in the ER and that Cd2+ does not inhibit disulphide bond formation (which could account for the lack of response in the case of Zn2+ and Hg2+) but perturbs calcium metabolism. Cd2+ activates the calcium channel Cch1/Mid1 under low external Ca2+, which also contributes to Cd2+ entry into the cell [93]; the protective effect of Ca2+ may be the result of competitive uptake between the two cations at the plasma membrane. In this line of evidence, it was shown that excess concentration of extracellular Ca2+ attenuates the Cd2+-induced ER stress [94]. It was

**Figure 5.** Cd2+-induced [Ca2+]cyt elevations mediate cell adaptation or cell death under Cd2+ stress. A. In normal (WT, wild type) cells, surplus Cd2+ induces Ca2+ entry via Cch1p/Mid1p channel, then [Ca2+]cyt is rapidly restored to low levels by the action of vacuolar Pmc1p and Vcx1p, allowing adaptation to high Cd2+. B. Cells lacking Cch1p or Mid1p (knock-out mutants *cch1Δ* or *mid1Δ*) die under Cd2+ stress, as Ca2+ does not enter the cell in sufficient quantity to signal the Cd2+ excess. C. Cells lacking both Pmr1p and Vcx1p (double knock-out mutant *pmr1Δ vcx1Δ*) die under Cd2+ stress, as [Ca2+] cyt cannot be rapidly restored to the low physiological levels [23].

determined that divalent Cd2+ and Ca2+ have very similar physical properties, with ionic radii of Ca2+ (0.97 Å) and Cd2+ (0.99 Å) giving similar charge/radius ratios, meaning that these ions are able to exert strong electrostatic forces on biological macromolecules [95]. Under such circumstances, the Cd2+-induced aequorin luminescence observed could also be the result of aequorin binding to Cd2+ instead of Ca2+. This was not the case though: when measuring the Cd2+ accumulation in yeast cells, it was revealed that the Cd2+-induced aequorin luminescence occurred significantly faster than the Cd2+ uptake, indicating that the luminescence produced was the result of increase in [Ca2+]cyt [23].
