**4. Calcium signalling and mitochondrial OXPHOS physiology**

Calcium (Ca2+) is one of the most common second messengers in intracellular signalling networks. Periodic fluctuations in cytosolic calcium concentration ([Ca2+]cyt) is driven by electrical activation of voltage-gated Ca2+ channels (VGCC) or by agonist stimulation of plasma membrane receptors and the subsequent formation of Ca2+-mobilizing second messengers, such as inositol 1,4,5-trisphosphate (IP3). IP3 binds to its receptor the IP3R (inositol 1,4,5-trisphosphate) on the endoplasmic reticulum (ER) membrane leading to Ca2+ release from the ER to the cytosol. In excitable cells, Ca2+ release from the ER occurs also through ryanodine receptors (RyR) that function as Ca2+-activated Ca2+ channels which further amplify Ca2+ signals originating from other sources.

The frequency, amplitude and/or duration of cytosolic [Ca2+]cyt spikes can be detected and decoded by downstream Ca2+-sensitive proteins providing a versatile pathway for extracellular stimuli to exert control over a wide range of metabolic pathways (Berridge et al., 2000).

Complex buffering systems that include multiple Ca2+-buffering proteins, ATP-dependent Ca2+ pumps (SERCA (sarco-endoplasmic Reticulum Ca2+ ATPase) accumulating Ca2+ from the cytosol to the ER, and PMCA (Plasma membrane Ca2+ ATPase) extruding Ca2+ from cytosol to the extracellular space), and the sodium-Ca2+ exchanger (Na+/Ca2+), work together to restore [Ca2+] back to resting levels. Mitochondria also play an important role in shaping Ca2+ signals by utilizing potent mitochondrial Ca2+ uptake mechanisms. Ca2+

Mitochondrial Calcium Signalling: Role in Oxidative Phosphorylation Diseases 39

Ca2+. They showed that receptor-activated Ca2+ signals caused rapid and large Ca2+ signals in the mitochondrial matrix (mechanisms of mitochondrial Ca2+ influx and efflux are

Mitochondrial Ca2+ uptake is dependent on the strong driving force ensured by their membrane potential (*-*180 mV, negative inside) built by the respiratory chain (for review see (Bianchi et al., 2004)). It has been assumed that [Ca2+]cyt far exceeding the micromolar range is required for net Ca2+ uptake, however, such [Ca2+]cyt values have not been observed experimentally in the bulk cytoplasm. Ca2+ diffusion in the cytoplasm is also controlled by protein binding (Allbritton et al., 1992). Thus, local Ca2+ transients with amplitudes far exceeding those measured over the global cytoplasm are confined in cytosolic microdomains at the mouth of Ca2+ channels beneath the plasma membrane or ER internal store. This concept was consolidated by the demonstration that mitochondria, forming a complex cytoplasmic tubulovesicular system (Tinel et al., 1999), are frequently apposed to the smooth as well as the rough ER. These contact points, have been observed in several cell types by means of electron microscopy or tomography (Mannella et al., 1998). The experiments by Rosario Rizzuto and Tulio Pozzan definitively demonstrated that Ca2+ released through IP3R in these microdomains, induce supramicromolar, or even

Accordingly, the group of György Hajnoczky demonstrates that maximal activation of mitochondrial Ca2+ uptake is evoked by IP3-induced perimitochondrial [Ca2+] elevations, which appear to reach values >20-fold higher than the global increases of [Ca2+]cyt. Incremental doses of IP3 elicited [Ca2+]mit elevations that followed the quantal pattern of Ca2+ mobilization, even at the level of individual mitochondria. These results and others by the same group allow concluding that each mitochondrial Ca2+ uptake site faces multiple IP3R, a concurrent activation of which is required for optimal activation of mitochondrial Ca2+ uptake (Csordas et al., 1999; Hajnoczky et al., 1995) and reviewed in (Csordas et al., 2006). Targeting aequorin to the outer surface of the IMM in HeLa cells made the measurement of [Ca2+] in the mitochondrial intermembrane space possible. After stimulation with histamine [Ca2+] rose in the intermembrane space to significantly higher values than in the global cytosol (Rizzuto et al., 1998). This observation has given a strong support to the concept that net mitochondrial Ca2+ uptake occurs from high-Ca2+

The existence of physical support for the ER–mitochondrial interface has been indicated by co-sedimentation of ER particles with mitochondria and electron microscopic observations of close associations between mitochondria and ER vesicles (Mannella et al., 1998; Meier et al., 1981; Shore & Tata, 1977). At these sites the shortest ER-OMM distance varies from 10 nm to 100 nm. In cells exposed to ER stress (serum starvation, tunicamycin) an increase in the ER–mitochondrial interface has been observed (Csordas et al., 2006). Also, coupling of the two organelles with a fusion protein increased the ER–mitochondria interface area, reduced the ER–mitochondrial distance to about 6 nm and greatly facilitated the transfer of cytosolic Ca2+ signal into the mitochondria of RBL-2H3 cells (Csordas et al., 2006). Accordingly, our team showed that the truncated variant of the sarco-endoplasmic

**4.2 Mechanisms of mitochondrial calcium influx and efflux** 

**4.2.1 Mechanisms of mitochondrial calcium influx** 

submillimolar Ca2+ signals (Rizzuto et al., 1993).

peri-mitochondrial microdomains.

detailed below).

uptake into mitochondria plays an important role in cellular physiology by stimulating mitochondrial metabolism and increasing mitochondrial energy production (Duchen, 1992). However, excessive Ca2+ uptake into mitochondria can lead to opening of a permeability transition pore (PTP) and apoptosis.

## **4.1 Interplay between Ca2+ and OXPHOS**

Mitochondrial bioenergetics and Ca2+ shaping are mutually regulated. Indeed, on the one hand, mitochondria Ca2+ accumulation enables the activity of OXPHOS and ATP production; on the other hand, mitochondrial ATP favours the effective functioning of the two major Ca2+ pumps PCMA and SERCA and actively participates in shaping cytosolic Ca2+ signals (Figure 1 A and B).

One important target for Ca2+ signals is the activation of mitochondrial oxidative metabolism and the consequent increase in the formation of ATP. Studies performed in 1960-1970 led to the demonstration that four mitochondrial dehydrogenases are activated by Ca2+ ions. These are FAD-glycerol phosphate dehydrogenase, pyruvate dehydrogenase, NAD+-isocitrate dehydrogenase and α-ketoglutarate dehydrogenase. FAD-glycerol phosphate dehydrogenase is located on the outer surface of the inner mitochondrial membrane and is influenced by changes in cytoplasmic Ca2+ ions concentrations. The other three enzymes are located within mitochondria and are regulated by matrix Ca2+ ions concentration. The effects of Ca2+ ions on FAD-isocitrate dehydrogenase involve binding to an EF-hand binding motif within this enzyme, leading to lowering of the *K*m for glycerol phosphate very substantially (reviewed in (Denton, 2009)). Mitochondrial Ca2+ ions bind also directly to NAD+-isocitrate dehydrogenase and α-ketoglutarate dehydrogenase to decrease the *K*m for their respective substrates, whereas an increase in the dephosphorylated and active form of pyruvate dehydrogenase is regulated by a Ca2+-sensitive phosphatase (Bulos et al., 1984; Denton & Hughes, 1978; Denton et al., 1972, 1978, 1996; McCormack et al., 1990; McCormack & Denton, 1979; Robb-Gaspers et al., 1998). Extramitochondrial Ca2+ regulates the glutamate-dependent state 3 respiration by the supply of glutamate to mitochondria via aralar, a mitochondrial glutamate/aspartate carrier (Gellerich et al., 2010).

A very recent finding suggests a novel paradigm in which the transcription of genes for mitochondrial enzymes that produce ATP and the genes that consume ATP is coordinately regulated by the same transcription factors (Watanabe et al., 2011). Thus, TFAM and TFB2M, recognized as mtDNA-specific transcription factors, were shown to regulate transcription of the SERCA2 gene (Watanabe et al., 2011).

It was also demonstrated that metabolites generated during energy production may influence IP3R-mediated Ca2+ dynamics. Indeed, it was shown that reduced Nicotinamide adenine dinucleotide selectively stimulates the release of Ca2+ mediated by IP3R (Kaplin et al., 1996). Another evidence of communication between cellular metabolism and Ca2+ signalling was reported recently by Bakowski and Parekh who showed that pyruvate, the precursor substrate for the Krebs cycle, directly increases the native ICRAC (store operated Ca2+ influx channels at the plasma membrane) by reducing inactivation of the channel, thereby coupling oxidation of glucose and its own metabolism in the mitochondria to Ca2+ influx by the CRAC channel (Bakowski & Parekh, 2007).

In addition to serving as a target of Ca2+ signalling, the uptake of Ca2+ by mitochondria has important feedback effects to shape cytosolic Ca2+ signals. Rosario Rizzuto and collaborators (Rizzuto et al., 1993) were the first to make direct *in situ* measurements of mitochondrial

uptake into mitochondria plays an important role in cellular physiology by stimulating mitochondrial metabolism and increasing mitochondrial energy production (Duchen, 1992). However, excessive Ca2+ uptake into mitochondria can lead to opening of a permeability

Mitochondrial bioenergetics and Ca2+ shaping are mutually regulated. Indeed, on the one hand, mitochondria Ca2+ accumulation enables the activity of OXPHOS and ATP production; on the other hand, mitochondrial ATP favours the effective functioning of the two major Ca2+ pumps PCMA and SERCA and actively participates in shaping cytosolic

One important target for Ca2+ signals is the activation of mitochondrial oxidative metabolism and the consequent increase in the formation of ATP. Studies performed in 1960-1970 led to the demonstration that four mitochondrial dehydrogenases are activated by Ca2+ ions. These are FAD-glycerol phosphate dehydrogenase, pyruvate dehydrogenase, NAD+-isocitrate dehydrogenase and α-ketoglutarate dehydrogenase. FAD-glycerol phosphate dehydrogenase is located on the outer surface of the inner mitochondrial membrane and is influenced by changes in cytoplasmic Ca2+ ions concentrations. The other three enzymes are located within mitochondria and are regulated by matrix Ca2+ ions concentration. The effects of Ca2+ ions on FAD-isocitrate dehydrogenase involve binding to an EF-hand binding motif within this enzyme, leading to lowering of the *K*m for glycerol phosphate very substantially (reviewed in (Denton, 2009)). Mitochondrial Ca2+ ions bind also directly to NAD+-isocitrate dehydrogenase and α-ketoglutarate dehydrogenase to decrease the *K*m for their respective substrates, whereas an increase in the dephosphorylated and active form of pyruvate dehydrogenase is regulated by a Ca2+-sensitive phosphatase (Bulos et al., 1984; Denton & Hughes, 1978; Denton et al., 1972, 1978, 1996; McCormack et al., 1990; McCormack & Denton, 1979; Robb-Gaspers et al., 1998). Extramitochondrial Ca2+ regulates the glutamate-dependent state 3 respiration by the supply of glutamate to mitochondria via aralar, a mitochondrial glutamate/aspartate carrier (Gellerich et al., 2010). A very recent finding suggests a novel paradigm in which the transcription of genes for mitochondrial enzymes that produce ATP and the genes that consume ATP is coordinately regulated by the same transcription factors (Watanabe et al., 2011). Thus, TFAM and TFB2M, recognized as mtDNA-specific transcription factors, were shown to regulate transcription of

It was also demonstrated that metabolites generated during energy production may influence IP3R-mediated Ca2+ dynamics. Indeed, it was shown that reduced Nicotinamide adenine dinucleotide selectively stimulates the release of Ca2+ mediated by IP3R (Kaplin et al., 1996). Another evidence of communication between cellular metabolism and Ca2+ signalling was reported recently by Bakowski and Parekh who showed that pyruvate, the precursor substrate for the Krebs cycle, directly increases the native ICRAC (store operated Ca2+ influx channels at the plasma membrane) by reducing inactivation of the channel, thereby coupling oxidation of glucose and its own metabolism in the mitochondria to Ca2+

In addition to serving as a target of Ca2+ signalling, the uptake of Ca2+ by mitochondria has important feedback effects to shape cytosolic Ca2+ signals. Rosario Rizzuto and collaborators (Rizzuto et al., 1993) were the first to make direct *in situ* measurements of mitochondrial

transition pore (PTP) and apoptosis.

Ca2+ signals (Figure 1 A and B).

**4.1 Interplay between Ca2+ and OXPHOS** 

the SERCA2 gene (Watanabe et al., 2011).

influx by the CRAC channel (Bakowski & Parekh, 2007).

#### **4.2 Mechanisms of mitochondrial calcium influx and efflux 4.2.1 Mechanisms of mitochondrial calcium influx**

Mitochondrial Ca2+ uptake is dependent on the strong driving force ensured by their membrane potential (*-*180 mV, negative inside) built by the respiratory chain (for review see (Bianchi et al., 2004)). It has been assumed that [Ca2+]cyt far exceeding the micromolar range is required for net Ca2+ uptake, however, such [Ca2+]cyt values have not been observed experimentally in the bulk cytoplasm. Ca2+ diffusion in the cytoplasm is also controlled by protein binding (Allbritton et al., 1992). Thus, local Ca2+ transients with amplitudes far exceeding those measured over the global cytoplasm are confined in cytosolic microdomains at the mouth of Ca2+ channels beneath the plasma membrane or ER internal store. This concept was consolidated by the demonstration that mitochondria, forming a complex cytoplasmic tubulovesicular system (Tinel et al., 1999), are frequently apposed to the smooth as well as the rough ER. These contact points, have been observed in several cell types by means of electron microscopy or tomography (Mannella et al., 1998). The experiments by Rosario Rizzuto and Tulio Pozzan definitively demonstrated that Ca2+ released through IP3R in these microdomains, induce supramicromolar, or even submillimolar Ca2+ signals (Rizzuto et al., 1993).

Accordingly, the group of György Hajnoczky demonstrates that maximal activation of mitochondrial Ca2+ uptake is evoked by IP3-induced perimitochondrial [Ca2+] elevations, which appear to reach values >20-fold higher than the global increases of [Ca2+]cyt. Incremental doses of IP3 elicited [Ca2+]mit elevations that followed the quantal pattern of Ca2+ mobilization, even at the level of individual mitochondria. These results and others by the same group allow concluding that each mitochondrial Ca2+ uptake site faces multiple IP3R, a concurrent activation of which is required for optimal activation of mitochondrial Ca2+ uptake (Csordas et al., 1999; Hajnoczky et al., 1995) and reviewed in (Csordas et al., 2006). Targeting aequorin to the outer surface of the IMM in HeLa cells made the measurement of [Ca2+] in the mitochondrial intermembrane space possible. After stimulation with histamine [Ca2+] rose in the intermembrane space to significantly higher values than in the global cytosol (Rizzuto et al., 1998). This observation has given a strong support to the concept that net mitochondrial Ca2+ uptake occurs from high-Ca2+ peri-mitochondrial microdomains.

The existence of physical support for the ER–mitochondrial interface has been indicated by co-sedimentation of ER particles with mitochondria and electron microscopic observations of close associations between mitochondria and ER vesicles (Mannella et al., 1998; Meier et al., 1981; Shore & Tata, 1977). At these sites the shortest ER-OMM distance varies from 10 nm to 100 nm. In cells exposed to ER stress (serum starvation, tunicamycin) an increase in the ER–mitochondrial interface has been observed (Csordas et al., 2006). Also, coupling of the two organelles with a fusion protein increased the ER–mitochondria interface area, reduced the ER–mitochondrial distance to about 6 nm and greatly facilitated the transfer of cytosolic Ca2+ signal into the mitochondria of RBL-2H3 cells (Csordas et al., 2006). Accordingly, our team showed that the truncated variant of the sarco-endoplasmic

Mitochondrial Calcium Signalling: Role in Oxidative Phosphorylation Diseases 41

increased mitochondrial Ca2+ elevations and the contrary is observed upon UCP2/3 depletion. In addition, mice lacking UCP2 exhibited a reduced sensitivity to the Ca2+ uptake inhibitor ruthenium red. However, these findings were disputed by another study that reported normal mitochondrial Ca2+ uptake in mice genetically ablated for UCP2 and UCP3 (Brookes et al., 2008). Furthermore, it was recently showed that UCP3 modulates the activity of sarco/endoplasmic reticulum Ca2+ ATPases by decreasing mitochondrial ATP production (De Marchi et al., 2011). The mitochondrial Ca2+ alterations associated with changes in UCP3 levels therefore reflect the exposure of mitochondria to abnormal cytosolic Ca2+ concentrations and do not reflect changes in MCU activity. These data indicate that UCP3 is not the mitochondrial Ca2+ uniporter. In 2009, Jiang and collaborators identified the leucine zipper EF hand containing transmembrane protein 1(Letm1) as a molecule that regulate both mitochondrial Ca2+ and H+ concentrations (Jiang et al., 2009). Letm1 was reported to be a high-affinity mitochondrial Ca2+/H+ exchanger able to import Ca2+ at low (i.e. sub-micromolar) cytosolic concentrations into energized mitochondria. Earlier studies had however linked Letm1 to mitochondrial K+/H+ exchange and to the maintenance of ionic mitochondrial balance, the integrity of the mitochondrial network and cell viability (Dimmer et al., 2008; Nowikovsky et al., 2004). The high-affinity of Letm1 for Ca2+ and its postulated 1Ca2+/1 H+ stoichiometry are at odds with the known properties of the MCU. Thus, Letm1 is not the dominant mechanism of mitochondrial Ca2+ uptake. Instead, Letm1 might contribute to an alternate mode of mitochondrial Ca2+ uptake, known as rapid mode of uptake (RaM), that was first reported in isolated rat liver mitochondria by Gunter's group. It was reported that mitochondrial Ca2+ sequestration via a the RaM occurred at the beginning of each pulse and was followed by a slower Ca2+ uptake characteristic of the MCU (Sparagna et al., 1995; Szabadkai et al., 2001). The implications of the coexistence of low and high-affinity modes of Ca2+ uptake have been

In 2010, Palmer and Mootha reported that a new mitochondrial EF hand protein MICU1 (for mitochondrial Ca2+ uptake 1) was required for high capacity mitochondrial Ca2+ uptake, and proposed that MICU1 acts as a Ca2+ sensor that controls the entry of Ca2+ across the uniporter (Perocchi et al., 2010). Building up on this discovery, the same group and another simultaneously identified the mitochondrial Ca2+ uniporter (Baughman et al., 2011; De Stefani et al., 2011). Using *in silico* analysis combined with phylogenetic profiling and analysis of RNA and protein co-expressed with MICU1, the group of Vamsi Mootha isolated a novel protein that co-immunoprecipitated with MICU1 (Baughman et al., 2011). Using the same database, the group of Rosario Rizzuto independently identified the same protein. From the 14 proteins characterized by two or more transmembrane domains and known to exhibit or lack uniport activity domains, these authors identified a protein with a highly conserved domain encompassing two transmembrane regions separated by a loop bearing acidic residues. Functional analysis confirmed that this protein behaves as expected for the mitochondrial uniporter, and it was therefore assigned the defining name of MCU. Mitochondrial Ca2+ uptake was strongly reduced by MCU silencing in cultured cells and in purified mouse liver mitochondria, whereas MCU overexpression enhanced ruthenium redsensitive mitochondrial Ca2+ uptake in intact and permeabilized cells (De Stefani et al., 2011). The MCU is a 45 kDa protein that can forms oligomers (Baughman et al., 2011). Both studies mapped the MCU to the inner mitochondrial membrane, but disagreed on whether the N and C termini face the matrix of the inter-membrane space (Baughman et al., 2011; De

recently reviewed (Santo-Domingo & Demaurex, 2010).

reticulum Ca2+-ATPase 1 (S1T) is induced under ER stress conditions. S1T is localized in the ER-mitochondria microdomains, increases number of ER-mitochondria contact sites, and inhibits mitochondria movements thus determining a privileged Ca2+ transfer from the ER to mitochondria leading to the activation of the mitochondrial apoptotic pathway (Chami et al., 2008).

Mitochondrial fission and fusion is another essential phenomenon for maintaining the metabolic function of these organelles as well as regulating their roles in cell signalling (Tatsuta & Langer, 2008; Yaffe, 1999; Chan, 2006). Changes in the relative rates of fusion and fission alter the overall morphology of the mitochondria affecting the function of the organelles both as regulators of survival/apoptosis and in Ca2+ handling. It has been shown that fusion is blocked (Karbowski & Youle, 2003) and mitochondria become fragmented during apoptosis (Frank et al., 2001). However, enhanced fission alone does not induce apoptosis and has even been shown to protect against Ca2+-dependent apoptosis by preventing the propagation of harmful Ca2+ waves through the mitochondrial reticulum (Szabadkai et al., 2004).

The outer mitochondrial membrane is permeable to solutes and the inner mitochondrial membrane is impermeable to solutes that harbor the respiratory chain complexes. As described in chapter 1, the respiratory chain pumps protons against their concentration gradient from the matrix of the mitochondrion into the inter-membrane space, generating an electrochemical gradient in the form of a negative inner membrane potential and of a pH gradient, the matrix being more alkaline than the cytosol (Bernardi et al., 1999; Poburko et al., 2011).

Ca2+ import across the outer mitochondrial membrane (OMM) occurs through the voltagedependent anion channels (VDAC) (Simamura et al., 2008). VDAC is as a large voltagegated channel, fully opened with high-conductance and weak anion-selectivity at low transmembrane potentials (< 20–30 mV), but switching to cation selectivity and lower conductance at higher potentials (Colombini, 2009; Shoshan-Barmatz et al., 2010). The precise mechanisms of VDAC conductance are however still under debate.

Ca2+ import across the inner mitochondrial membrane (IMM) occurs through a Ca2+ selective channel known as the mitochondrial Ca2+ uniporter (MCU) (Kirichok et al., 2004). Electrophysiological recordings of mitoplasts, small vesicles of inner mitochondrial membrane, revealed that the MCU is a highly Ca2+-selective inward-rectifying ion channel (Kirichok et al., 2004). The MCU has a relatively low Ca2+ affinity (Kd 10 μM in permeabilized cells (Bernardi, 1999)). The activity of the MCU had been known for decades to be inhibited by ruthenium red and its derivative Ru360 (Vasington et al., 1972), but its molecular identity has only been unraveled very recently. It has been reported recently that the process of Ca2+ accumulation undergoes complex regulation by Ca2+ itself. Thus mitochondrial uptake of Ca2+ was significantly reduced by inhibitors of calmodulin, suggesting that a Ca2+–calmodulin-mediated process is necessary for activation of the uniporter but Ca2+ also appeared to inhibit its own uptake. However, in contrast to the sensitization of mitochondrial Ca2+ uptake, the Ca2+-dependent inactivation was not sensitive to calmodulin blockers (Moreau & Parekh, 2008).

In recent years, several molecules have been proposed to be either an essential or an accessory component of the MCU. In 2007, the uncoupling proteins (UCP) 2 and 3 (Trenker et al., 2007) were proposed to be essential for the MCU. Indeed, UCP2/3 overexpression

reticulum Ca2+-ATPase 1 (S1T) is induced under ER stress conditions. S1T is localized in the ER-mitochondria microdomains, increases number of ER-mitochondria contact sites, and inhibits mitochondria movements thus determining a privileged Ca2+ transfer from the ER to mitochondria leading to the activation of the mitochondrial apoptotic pathway

Mitochondrial fission and fusion is another essential phenomenon for maintaining the metabolic function of these organelles as well as regulating their roles in cell signalling (Tatsuta & Langer, 2008; Yaffe, 1999; Chan, 2006). Changes in the relative rates of fusion and fission alter the overall morphology of the mitochondria affecting the function of the organelles both as regulators of survival/apoptosis and in Ca2+ handling. It has been shown that fusion is blocked (Karbowski & Youle, 2003) and mitochondria become fragmented during apoptosis (Frank et al., 2001). However, enhanced fission alone does not induce apoptosis and has even been shown to protect against Ca2+-dependent apoptosis by preventing the propagation of harmful Ca2+ waves through the mitochondrial reticulum

The outer mitochondrial membrane is permeable to solutes and the inner mitochondrial membrane is impermeable to solutes that harbor the respiratory chain complexes. As described in chapter 1, the respiratory chain pumps protons against their concentration gradient from the matrix of the mitochondrion into the inter-membrane space, generating an electrochemical gradient in the form of a negative inner membrane potential and of a pH gradient, the matrix being more alkaline than the cytosol (Bernardi et al., 1999; Poburko et

Ca2+ import across the outer mitochondrial membrane (OMM) occurs through the voltagedependent anion channels (VDAC) (Simamura et al., 2008). VDAC is as a large voltagegated channel, fully opened with high-conductance and weak anion-selectivity at low transmembrane potentials (< 20–30 mV), but switching to cation selectivity and lower conductance at higher potentials (Colombini, 2009; Shoshan-Barmatz et al., 2010). The

Ca2+ import across the inner mitochondrial membrane (IMM) occurs through a Ca2+ selective channel known as the mitochondrial Ca2+ uniporter (MCU) (Kirichok et al., 2004). Electrophysiological recordings of mitoplasts, small vesicles of inner mitochondrial membrane, revealed that the MCU is a highly Ca2+-selective inward-rectifying ion channel (Kirichok et al., 2004). The MCU has a relatively low Ca2+ affinity (Kd 10 μM in permeabilized cells (Bernardi, 1999)). The activity of the MCU had been known for decades to be inhibited by ruthenium red and its derivative Ru360 (Vasington et al., 1972), but its molecular identity has only been unraveled very recently. It has been reported recently that the process of Ca2+ accumulation undergoes complex regulation by Ca2+ itself. Thus mitochondrial uptake of Ca2+ was significantly reduced by inhibitors of calmodulin, suggesting that a Ca2+–calmodulin-mediated process is necessary for activation of the uniporter but Ca2+ also appeared to inhibit its own uptake. However, in contrast to the sensitization of mitochondrial Ca2+ uptake, the Ca2+-dependent inactivation was not

In recent years, several molecules have been proposed to be either an essential or an accessory component of the MCU. In 2007, the uncoupling proteins (UCP) 2 and 3 (Trenker et al., 2007) were proposed to be essential for the MCU. Indeed, UCP2/3 overexpression

precise mechanisms of VDAC conductance are however still under debate.

sensitive to calmodulin blockers (Moreau & Parekh, 2008).

(Chami et al., 2008).

(Szabadkai et al., 2004).

al., 2011).

increased mitochondrial Ca2+ elevations and the contrary is observed upon UCP2/3 depletion. In addition, mice lacking UCP2 exhibited a reduced sensitivity to the Ca2+ uptake inhibitor ruthenium red. However, these findings were disputed by another study that reported normal mitochondrial Ca2+ uptake in mice genetically ablated for UCP2 and UCP3 (Brookes et al., 2008). Furthermore, it was recently showed that UCP3 modulates the activity of sarco/endoplasmic reticulum Ca2+ ATPases by decreasing mitochondrial ATP production (De Marchi et al., 2011). The mitochondrial Ca2+ alterations associated with changes in UCP3 levels therefore reflect the exposure of mitochondria to abnormal cytosolic Ca2+ concentrations and do not reflect changes in MCU activity. These data indicate that UCP3 is not the mitochondrial Ca2+ uniporter. In 2009, Jiang and collaborators identified the leucine zipper EF hand containing transmembrane protein 1(Letm1) as a molecule that regulate both mitochondrial Ca2+ and H+ concentrations (Jiang et al., 2009). Letm1 was reported to be a high-affinity mitochondrial Ca2+/H+ exchanger able to import Ca2+ at low (i.e. sub-micromolar) cytosolic concentrations into energized mitochondria. Earlier studies had however linked Letm1 to mitochondrial K+/H+ exchange and to the maintenance of ionic mitochondrial balance, the integrity of the mitochondrial network and cell viability (Dimmer et al., 2008; Nowikovsky et al., 2004). The high-affinity of Letm1 for Ca2+ and its postulated 1Ca2+/1 H+ stoichiometry are at odds with the known properties of the MCU. Thus, Letm1 is not the dominant mechanism of mitochondrial Ca2+ uptake. Instead, Letm1 might contribute to an alternate mode of mitochondrial Ca2+ uptake, known as rapid mode of uptake (RaM), that was first reported in isolated rat liver mitochondria by Gunter's group. It was reported that mitochondrial Ca2+ sequestration via a the RaM occurred at the beginning of each pulse and was followed by a slower Ca2+ uptake characteristic of the MCU (Sparagna et al., 1995; Szabadkai et al., 2001). The implications of the coexistence of low and high-affinity modes of Ca2+ uptake have been recently reviewed (Santo-Domingo & Demaurex, 2010).

In 2010, Palmer and Mootha reported that a new mitochondrial EF hand protein MICU1 (for mitochondrial Ca2+ uptake 1) was required for high capacity mitochondrial Ca2+ uptake, and proposed that MICU1 acts as a Ca2+ sensor that controls the entry of Ca2+ across the uniporter (Perocchi et al., 2010). Building up on this discovery, the same group and another simultaneously identified the mitochondrial Ca2+ uniporter (Baughman et al., 2011; De Stefani et al., 2011). Using *in silico* analysis combined with phylogenetic profiling and analysis of RNA and protein co-expressed with MICU1, the group of Vamsi Mootha isolated a novel protein that co-immunoprecipitated with MICU1 (Baughman et al., 2011). Using the same database, the group of Rosario Rizzuto independently identified the same protein. From the 14 proteins characterized by two or more transmembrane domains and known to exhibit or lack uniport activity domains, these authors identified a protein with a highly conserved domain encompassing two transmembrane regions separated by a loop bearing acidic residues. Functional analysis confirmed that this protein behaves as expected for the mitochondrial uniporter, and it was therefore assigned the defining name of MCU. Mitochondrial Ca2+ uptake was strongly reduced by MCU silencing in cultured cells and in purified mouse liver mitochondria, whereas MCU overexpression enhanced ruthenium redsensitive mitochondrial Ca2+ uptake in intact and permeabilized cells (De Stefani et al., 2011). The MCU is a 45 kDa protein that can forms oligomers (Baughman et al., 2011). Both studies mapped the MCU to the inner mitochondrial membrane, but disagreed on whether the N and C termini face the matrix of the inter-membrane space (Baughman et al., 2011; De

Mitochondrial Calcium Signalling: Role in Oxidative Phosphorylation Diseases 43

primary cells in response to physiological activators that dictate cytosolic Ca2+ has remained a major challenge. Yet, opening of the PTP is often thought to be associated with pathophysiological processes (for reviews see (Hajnoczky et al., 2006; Rizzuto et al., 2003)). In these scenarios, activation of the PTP leads to respiratory inhibition, and thus ATP depletion, and the release of mitochondrial Ca2+ stores and apoptotic activators, ultimately resulting in cell death (Bernardi et al., 1999; Di Lisa & Bernardi, 2009). These have led to the idea that opening of the PTP by elevated mitochondrial Ca2+ is a terminal, pathologic event. However, it has been reported recently that CyPD-dependent PTP may participate in non-

The direct consequences of OXPHOS defects include alteration of mitochondrial membrane potential, ATP/ADP ratio, ROS production and mitochondrial Ca2+ homeostasis. The varied biochemical changes that occur in cases of OXPHOS deficiencies have a direct effect on cellular functions. Yet, they are also key underlying mediators of the (retrograde) communication between the mitochondrion and the nucleus, which results in specific gene expression of both nuclear and mitochondrial genomes (see review (Reinecke et al., 2009)). We will review in this chapter only Ca2+ deregulation in OXPHOS. We will discuss the consequences of such deregulation on mitochondrial function and the cross regulation between Ca2+ and bioenergetics in the development of cellular pathology. We summarized in Table 1 the alterations of subcellular Ca2+ signals in OXPHOS related diseases (Table 1). Decreased proton pumping due to respiratory chain defects can result in reduced mitochondrial membrane potential and proton gradient, which are used to generate ATP. Deregulation of the membrane potential secondary to a deficiency in the respiratory chain may modify the kinetics and/or accumulation capacity of Ca2+ in the mitochondria, with possible consequences not only at the level of respiratory chain function (loop effect) and of the mitochondria in general, but also at the level of the ER function, which is largely dependent on Ca2+ concentrations, and at the level of cytosolic Ca2+ signalling, which plays a major role in regulating cell functions. Deficiencies of OXPHOS also result in other immediate and downstream metabolic, structural, and functional effects. These effects are closely associated with mitochondrial dysfunction. The nicotinamide dinucleotide (NAD) redox balance, which is converted to the reduced state in OXPHOS deficiencies, is a fundamental mediator of several biological processes, such as energy metabolism, Ca2+ homeostasis, cellular redox balance, immunological function, and gene expression (Munnich

It is important to mention that analyses of Ca2+ signalling targeting OXPHOS diseases are sporadic, partial and incomplete. This situation can be explained by : 1) the recent development of new techniques permitting detailed and specific subcellular Ca2+ analyses such as recombinant "aequorin" probes developed by the group headed by Professors Rizzuto and Pozzan, and the latest generation of GFP-based Ca2+ probes (camgaroos, cameleons and pericams) characterized by a great potential to analyse Ca2+ dynamics in mitochondria at the single cell level; 2) Absence of suitable "easy" study models (see chapter 3); and 3) the difficulty in the characterization of OXPHOS deficiencies (see

lethal Ca2+ homeostasis in cells and neurons (Barsukova et al., 2011).

**5. Calcium deregulation in OXPHOS diseases** 

& Rustin, 2001; Ying, 2008).

chapter 2-2).

Stefani et al., 2011). Mutations of conserved acidic residues within the short sequence linking the two transmembrane domains abrogated the ability of MCU to reconstitute mitochondrial Ca2+ uptake, whereas mutation of a nearby serine residue (S259) conferred resistance to Ru360, indicating that the acidic residues are required for Ca2+ uptake and that S259 is critical for MCU sensitivity to ruthenium red (Baughman et al., 2011). Finally, and most convincingly, expression of the purified protein in planar lipid bilayers was sufficient to reconstitute ion channel activity in solutions containing only Ca2+ (De Stefani et al., 2011). The currents were carried by a channel of small conductance (6–7 pS), fast opening/closing kinetics, and low opening probability, and were inhibited by ruthenium red, as expected for the MCU. Proteins mutated at two of the conserved acidic residues failed to generate Ca2+ currents when inserted into bilayers and acted as dominant negative when expressed in HeLa cells. These data clearly identified MCU as mitochondrial Ca2+ uniporter. In accordance to the notion that mitochondrial Ca2+ overload enhances the sensitivity to apoptosis, it was also demonstrated that cells overexpressing MCU were more sensitive to apoptosis after treatment with ceramide and H2O2 (De Stefani et al., 2011) (Figure 1B).

#### **4.2.2 Mechanisms of mitochondrial calcium efflux**

Compared to the MCU, the proteins that catalyze the efflux of Ca2+ from mitochondria have received much less attention. The extrusion of Ca2+ from mitochondria is coupled to the entry of Na+ across an electrogenic 1Ca+:3Na+ exchanger (Dash & Beard, 2008) that is inhibited by the benzothiazepine derivative CGP37157 ((Cox et al., 1993), and reviewed in (Bernardi, 1999)). The subsequent efflux of sodium ions by the mitochondrial 1Na+:1H+ exchanger (mNHE) eventually results in the entry of three protons into the matrix for each Ca2+ ion that leaves mitochondria. Ca2+ extrusion thus has a high energetic cost, as it dissipates the proton gradient generated by the respiratory chain (reviewed in (Bernardi, 1999)). The molecule catalyzing mitochondrial Na+/Ca2+ exchange has been recently identified as NCLX/NCKX6, a protein localized in mitochondrial cristae (Palty et al., 2010), whereas stomatin-like protein 2 (SLP-2), an inner membrane protein, was shown to negatively modulate the activity of the mitochondrial Na+/Ca2+ exchanger (Da Cruz et al., 2010). Functional evidence from knock-down and overexpression studies indicate that NCLX is an essential part of the mitochondrial sodium Ca2+ exchanger whereas SLP-2 is an accessory protein that negatively regulates mitochondrial Ca2+ extrusion (Figure 1B).

#### **4.2.3 Mitochondrial calcium overload: Activation of the permeability transition pore**

When mitochondrial Ca2+ loads exceed the buffering capacity of inner membrane exchangers, an additional pathway for Ca2+ efflux from mitochondria may exist through opening of the permeability transition pore (PTP). The PTP is a voltage-dependent, cyclosporin A (CsA)-sensitive, high-conductance channel of the inner mitochondrial membrane (for reviews, see (Bernardi et al., 2006; Rasola & Bernardi, 2007)). Indeed, the interplay between the rate of mitochondrial Ca2+ influx and efflux modulates mitochondrial matrix Ca2+, which in turn is widely considered to be a key factor for the regulation of the PTP open–closed transitions (Bernardi et al., 1999). Although opening of the PTP in response to Ca2+ has been documented in isolated mitochondria and permeabilized cells (Bernardi et al., 2006; Rasola & Bernardi, 2007), assessing opening of the PTP in intact neurons and other primary cells in response to physiological activators that dictate cytosolic Ca2+ has remained a major challenge. Yet, opening of the PTP is often thought to be associated with pathophysiological processes (for reviews see (Hajnoczky et al., 2006; Rizzuto et al., 2003)). In these scenarios, activation of the PTP leads to respiratory inhibition, and thus ATP depletion, and the release of mitochondrial Ca2+ stores and apoptotic activators, ultimately resulting in cell death (Bernardi et al., 1999; Di Lisa & Bernardi, 2009). These have led to the idea that opening of the PTP by elevated mitochondrial Ca2+ is a terminal, pathologic event. However, it has been reported recently that CyPD-dependent PTP may participate in nonlethal Ca2+ homeostasis in cells and neurons (Barsukova et al., 2011).
