**4.2. Cu2+**

to understand the role of calcium in preparing the yeast cell to resist the heavy metal attack

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

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

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

cyt elevations in *S. cerevisiae*,

are summarized in the following sections.

restore the low levels of [Ca2+]cyt [23] (**Figure 5C**).

cannot be rapidly restored to the low physiological levels [23].

[92]. External Cd2+ was shown to unequivocally induce the [Ca2+]

**4.1. Cd2+**

30 Calcium and Signal Transduction

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 Cu2+ and reduced Cu<sup>+</sup> . Studies correlating Ca2+ with Cu2+ toxicity in yeast are scarce, but it had been known that the inhibitory effect of Cu2+ on glucose-dependent H<sup>+</sup> efflux from *S. cerevisiae* 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 high Cu2+ depend predominantly on internal Ca2+ stores [24] (**Figure 6A**).

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 oxidative status. Moreover, it was revealed that Cu2+ and H<sup>2</sup> O2 acted in synergy to induce 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), probably because these metals are less redox-reactive than the Cu2+/Cu<sup>+</sup> couple (**Figure 6D**) under aerobic conditions [98].

#### **4.3. Mn2+**

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

by Fe2+ surplus. As in the case of Mn2+, excess Fe2+ did not elicit sudden elevations in [Ca2+]cyt upon exposure [23]. It had been reported that yeast strains lacking the components of the Cch1p/Mid1p plasma membrane channel were hypersensitive to Fe2+. When measuring the relative Ca2+ accumulation, it was noted that iron stress also increased the residual Ca2+ uptake in the *cch1Δ mid1Δ* double knockout mutant [8]. As the Ca2+ measurements in this study were done radiometrically, there must have been a considerable lag between application of the stimulus and Ca2+ measurement (unlike aequorin determinations, which allow Ca2+ detection simultaneously with stimulus application), and the mutant's sensitivity towards Fe2+ might have been caused by Ca2+ lingering in the cytosol, as in the case of

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

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

33

rapidly induce elevations in [Ca2+]cyt. In some cases, (Ni2+ and Co2+) exogenous Ca2+ alleviated the toxicity of the metal ions, but this effect was rather related to the inhibition of Co2+ or Ni2+

In this chapter, we attempted to highlight the studies made in *S. cerevisiae* which correlate the exposure to high concentrations of heavy metals with the Ca2+-mediated cellular responses. *S. cerevisiae* is a very good model to study the cell response to sudden changes of metal concentration in the environment; such studies were greatly facilitated by the ease of obtaining yeast cells expressing aequorin in the cytosol, thus allowing the realtime detection of [Ca2+]cyt fluctuations. By combining Ca2+ monitoring under metal stress with the genetic approaches that make use of mutants with perturbed heavy metal or Ca2+ homeostasis, important aspects related to cell adaptation or cell death under heavy metal stress have been elucidated. Using yeast cells expressing aequorin in the cytosol provides answers regarding the immediate Ca2+-mediated responses, which are crucial for deciding the cell fate. Nevertheless, to understand the Ca2+-mediated cell responses which occur at later phases, developing sensitive Ca2+ sensors targeted to specific compartments is still a

Ileana Cornelia Farcasanu\*, Claudia Valentina Popa and Lavinia Liliana Ruta

Department of Organic Chemistry, Biochemistry and Catalysis, Faculty of Chemistry,

\*Address all correspondence to: ileana.farcasanu@chimie.unibuc.ro

University of Bucharest, Bucharest, Romania

did not have the ability to

The surplus of heavy metals such as Ni2+, Co2+, Pb2+, Hg2+, and Ag<sup>+</sup>

Cd2+-sensitive mutants [23].

**4.5. Other metals**

uptake by Ca2+ [103].

**5. Concluding remarks**

desiderate for future studies.

**Author details**

**Figure 6.** Cu2+-induced [Ca2+] cyt elevations mediate cell adaptation or cell death under Cu2+ stress. A. In normal (WT, wild type) cells, surplus Cu2+ induces [Ca2+] cyt elevations as Ca2+ enters via Cch1p/Mid1p channel or is released from the vacuole via Yvc1p, in a positive feed-back. The normal low levels of [Ca2+] cyt are not rapidly restored as in the case of Cd2+-exposure, and the cells die. B. Cells lacking Cch1p (but not Mid1p) exhibit lower elevations in Cu2+-induced [Ca2+] cyt and are more tolerant to Cu2+ stress. C. Cells lacking Yvc1p (knock-out mutant *yvc1Δ*) exhibit very low elevations in Cu2+ induced [Ca2+] cyt and adapt easily to Cu2+ stress [24]. The cell behavior described in A-C is similar to the Ca2+-mediated response to oxidative stress [18], suggesting that the Cu2+-induced [Ca2+] cyt changes may be indirectly mediated by the formation of reactive oxygen species during copper shuffling between oxidative states Cu2+-Cu<sup>+</sup> (D).

manifested at several levels [41]. For example, high Mn2+ is controlled by calcineurin/Crz1pregulated Pmr1p and Pun1p [100]. Importantly, the tolerance of yeast cells to Mn2+ is related to both Pmr1p and Vcx1p [41, 64, 65, 101] two determinants of maintaining low [Ca2+]cyt by transporting the ions to the vacuole and Golgi/ER, respectively [60–63]. The Ca2+-dependent response to Mn2+ surplus seems to be induced not by external Mn2+, but by the cations accumulated inside the cell. For example, it was found that internal Mn2+ can be redistributed by calcium-stimulated vesicle trafficking [102].

#### **4.4. Fe2+**

Fe2+ toxicity can be the result of direct ionic effect, but the indirect effect of catalyzing Fenton reactions, in which highly reactive oxygen species arise, represents the main concern raised by Fe2+ surplus. As in the case of Mn2+, excess Fe2+ did not elicit sudden elevations in [Ca2+]cyt upon exposure [23]. It had been reported that yeast strains lacking the components of the Cch1p/Mid1p plasma membrane channel were hypersensitive to Fe2+. When measuring the relative Ca2+ accumulation, it was noted that iron stress also increased the residual Ca2+ uptake in the *cch1Δ mid1Δ* double knockout mutant [8]. As the Ca2+ measurements in this study were done radiometrically, there must have been a considerable lag between application of the stimulus and Ca2+ measurement (unlike aequorin determinations, which allow Ca2+ detection simultaneously with stimulus application), and the mutant's sensitivity towards Fe2+ might have been caused by Ca2+ lingering in the cytosol, as in the case of Cd2+-sensitive mutants [23].

#### **4.5. Other metals**

The surplus of heavy metals such as Ni2+, Co2+, Pb2+, Hg2+, and Ag<sup>+</sup> did not have the ability to rapidly induce elevations in [Ca2+]cyt. In some cases, (Ni2+ and Co2+) exogenous Ca2+ alleviated the toxicity of the metal ions, but this effect was rather related to the inhibition of Co2+ or Ni2+ uptake by Ca2+ [103].
