Calcium and bioenergetics: from endoplasmic reticulum to mitochondria

Abstract

Controlling metabolism throughout life is a necessity for living creatures, and perturbation of energy balance elicits disorders such as type-2 diabetes mellitus and cardiovascular disease. Ca²⁺ plays a key role in regulating energy generation. Ca²⁺ homeostasis of the endoplasmic reticulum (ER) lumen is maintained through the action of Ca²⁺ channels and the Ca²⁺ ATPase pump. Once released from the ER, Ca²⁺ is taken up by mitochondria where it facilitates energy metabolism. Mitochondrial Ca²⁺ serves as a key metabolic regulator and determinant of cell fate, necrosis, and/or apoptosis. Here, we focus on Ca²⁺ transport from the ER to mitochondria, and Ca²⁺-dependent regulation of mitochondrial energy metabolism.

Keywords:

• Ca²⁺ homeostasis
• receptor
• endoplasmic reticulum
• mitochondria
• energy metabolism

Introduction

The regulation and balance of cellular energy metabolism are fundamental requirements for all living organisms. Adenosine-5′-triphosphate (ATP) is generated by the catabolism of nutrients and consumption of oxygen in the mitochondria. When ATP is hydrolyzed, free energy is released to support the metabolic needs of the cell and maintain homeostatic processes.

The endoplasmic reticulum (ER), found in all eukaryotic cells, plays a fundamental role in protein synthesis, maturation, and sorting to their final destination. Additionally, the ER lumen is a major reservoir of intracellular Ca²⁺, and fluctuations in ER Ca²⁺ results in impaired movement of Ca²⁺ between the ER and Golgi (Ashby and Tepikin 2001), and impeded transport of small molecules across the nuclear pore (Greber and Gerace 1995). ER Ca²⁺ homeostasis and Ca²⁺ signaling are maintained by controlling Ca²⁺ release from the ER by the inositol 1,4,5-triphosphate receptor (InsP₃R) and ryanodine receptor (RyR). The sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (SERCA) pump is responsible for returning Ca²⁺ to the ER lumen. The ER and mitochondria form close contacts, and Ca²⁺ released from the ER via the InsP₃R is efficiently transported into the mitochondria where it supports ATP synthesis (Cardenas et al. 2010).

Mitochondrial Ca²⁺ homeostasis is an emerging field of research that includes study of the regulation of basic mitochondrial functions and cell fate (life and/or death). In this review, we have focused on the mechanisms responsible for transfer of Ca²⁺ from the InsP₃-sensitive ER to the mitochondria matrix and the effect of Ca²⁺ on mitochondrial metabolism.

Ca²⁺ homeostasis in the ER

The InsP₃R is one of two major Ca²⁺ channels that generates cell signaling-derived Ca²⁺ release from the ER lumen to the cytosol (Yule et al. 2010). This receptor is activated by InsP₃, a second messenger, as well as Ca²⁺, which is an allosteric modulator and plays an important role in shaping the InsP₃R-evoked Ca²⁺ response. Interestingly, Ca²⁺ enhances the probability that the InsP₃R will be open but never allows independent opening of the receptor in the absence of InsP₃ ligand. The RyR is the second ER Ca²⁺ channel, which is ubiquitously expressed in cells and shows high-conductance relative to other nonspecific cation channels. The mammalian genome includes three RyR isoforms. RyR1 is prominent in skeletal muscle, where it functions in excitation–contraction (EC) coupling and muscle contraction. RyR2 is predominantly expressed in cardiac muscle and is also the major form in brain tissue. Lastly, RyR3 exhibits a low level widespread expression pattern and is found in striated, smooth, and cardiac muscle, as well as in T lymphocytes and in the brain-specific regions for learning and memory (Hertle and Yeckel 2007). Furthermore, Ca²⁺ is the principal activator of all three RyR isoforms, however, each isoform displays different sensitivities to cytosolic Ca²⁺ (RyR1 > RyR2 > RyR3) (Fill and Copello 2002).

Uptake of cytosolic Ca²⁺ into the ER lumen is mediated by the SERCA pump via ATP hydrolysis. The SERCA2a isoform is expressed both in cardiac and slow skeletal muscle, whereas SERCA2b is ubiquitously expressed in non-muscle tissues, particularly in the brain (Carafoli and Brini 2000). In the lumen of the ER Ca²⁺ is stored bound to ER luminal Ca²⁺ buffering chaperones and folding enzymes (Michalak et al. 2009). Ca²⁺ also binds to the EF hand domain of stromal interacting molecule (STIM), a regulator of store-operated Ca²⁺ entry (SOCE) (Roos et al. 2005). Upon depletion of ER Ca²⁺ stores and disassociation of Ca²⁺ from STIM1, the protein undergoes oligomerization at sites immediately adjacent to the plasma membrane, and binds Orai, a SOCE channel (Hewavitharana et al. 2007). Thus, the STIM1–Orai complex SOCE refills ER stores with Ca²⁺ funneled from the extracellular space directly into the ER lumen (Lewis 2007; Prins et al. 2011). A large portion of the luminal ER Ca²⁺ is free (50–800 µM) (Bygrave and Benedetti 1996), and this allows for rapid diffusion of Ca²⁺ throughout the ER lumen. High-capacity Ca²⁺ buffering proteins such as calreticulin play a critical role in maintaining ER Ca²⁺ homeostasis (Michalak et al. 2009). Calreticulin is one of the Ca²⁺-buffering proteins in the ER lumen. The protein utilizes a carboxyl terminal acidic region as the high-capacity Ca²⁺-binding site to bind 25 mol of Ca²⁺ per mol of protein with low affinity (K _d=2 mM) (Baksh and Michalak 1991). Over 50% of Ca²⁺ stored in the ER lumen is bound to calreticulin (Nakamura et al. 2001), and up-regulation of calreticulin leads to increased amounts of Ca²⁺ in ER intracellular stores (Arnaudeau et al. 2002), whereas calreticulin-deficient cells have reduced Ca²⁺-storage capacity in the ER and delayed agonist-induced Ca²⁺ release (Guo et al. 2001; Michalak et al. 2009). Besides calreticulin, binding immunoglobulin protein (BiP), glucose-regulated protein 94 (GRP94), and protein disulfide isomerase (PDI) also take part in the regulation of ER Ca²⁺ homeostasis. BiP binds Ca²⁺ with a relatively low capacity (1–2 mol of Ca²⁺ per mol of protein), however, is responsible for as much as 25% of the total Ca²⁺ binding capacity of the ER (Lievremont et al. 1997). GRP94 is one of the most abundant Ca²⁺-buffering proteins of the ER with both low-affinity and high-capacity for Ca²⁺. GRP94 has 15 moderate-affinity sites (K _d=~2 µM) with low capacity (1 mol Ca²⁺ per mol of protein), and 11 low-affinity sites (K _d=~600 µM) with high capacity (10 mol of Ca²⁺ per mol of protein) (Argon and Simen 1999). As a Ca²⁺-buffering oxidoreductase in the ER lumen, PDI binds Ca²⁺ with a high capacity (19 mol Ca²⁺ per mol of protein) and weak affinity (K _d=2–5 mM) (Lebeche et al. 1994).

Mitochondrial Ca²⁺ uptake

Ca²⁺ uptake can be monitored using imaging techniques such as the Ca²⁺-sensitive photoprotein, aequorin (Rizzuto et al. 1992). This protein allows the selective measurement and monitoring of mitochondrial Ca²⁺ concentration and uptake. Using engineered Cameleon probes (Dcpv), Ca²⁺ hotspots have been directly visualized in intact cells, demonstrating the close apposition of mitochondria to ER Ca²⁺ channels and some types of plasma membrane Ca²⁺ channels (Giacomello et al. 2010). The mitochondrial outer membrane is highly permeable to ions and solutes compare with the mitochondrial inner membrane, which is ion impermeable (Kirichok et al. 2004). The voltage-dependent anion channel (VDAC) is clustered at ER/mitochondrial contact sites and it influences Ca²⁺ uptake by mitochondria (Rapizzi et al. 2002). High-speed single cell imaging has shown that up-regulation of VDAC triggers a significant rise in the peak Ca²⁺ concentration and reduces the delay between the cytosolic and mitochondrial upstroke (Rapizzi et al. 2002). Therefore, VDAC, located in the outer mitochondrial membrane, plays a critical role in the rapid transfer of the high Ca²⁺ microdomain from the outside of the mitochondria to internal mitochondrial space. Interestingly, VDAC shuttles between open and closed states (Tan and Colombini 2007). Ca²⁺ may also play a role in control of the conductance of VDAC (Tan and Colombini 2007), thus suggesting that mitochondrial Ca²⁺ uptake may be facilitated by a reduced barrier of the mitochondrial outer membrane during intracellular Ca²⁺ signaling.

In mitochondria-associated ER membrane (MAM), proteins within the ER are associated directly with proteins and lipids of the mitochondrial outer membrane. Isolation of MAM fractions has allowed the identification of proteins that might be important in ER/mitochondrial Ca²⁺ communication. For example, chaperones and proteins controlling the fusion and fission of mitochondria have been isolated from MAM fractions (Szabadkai et al. 2006). Glucose-regulated protein 75 (GRP75), a chaperone which assists with the refolding of newly imported proteins in the mitochondria matrix, was identified as a VDAC binding partner in a yeast two-hybrid screening. GRP75 mediated the interaction of VDAC1 with the InsP₃R1 at MAM, allowing the InsP₃R to facilitate mitochondria Ca²⁺ uptake (Szabadkai et al. 2006). The sigma-1 receptor is a Ca²⁺-sensitive ER chaperone that stabilizes InsP₃R when the Ca²⁺ concentration within the ER drops, and is another component of MAM that contributes to the maintenance of ER–mitochondria communications (Szabadkai et al. 2006).

Since most of the mitochondrial Ca²⁺ effectors are localized in the mitochondrial matrix, Ca²⁺ transport from the outside environment to the matrix through the highly permeable outer membrane and ion impermeable inner membrane is a key limiting factor. Major efforts have been made to elucidate the inner membrane Ca²⁺ transporters. Mitochondria Ca²⁺ uniporter (MCU) was identified, which is an electrophoretic system that has the properties of a highly selective ion channel (Kirichok et al. 2004), and allows Ca²⁺ to be accumulated down the electrical gradient established by the respiratory chain (Carafoli 2003). Mitochondrial Ca²⁺ uptake 1 (MICU1) was identified by screening for the expected properties of MCU. MICU1 is associated with the inner mitochondrial membrane and possesses 2 EF-hand Ca²⁺-binding sites as well as a single transmembrane stretch (Perocchi et al. 2010). In intact and permeabilized cells, silencing of MICU1 abolishes mitochondrial Ca²⁺ uptake without interfering with mitochondrial respiration or membrane potential (Perocchi et al. 2010). Indeed MICU1 is required for the metabolic coupling between cytosolic Ca²⁺ transients and activation of matrix dehydrogenase.

Cellular metabolism by mitochondria Ca²⁺

In the mitochondrial matrix, the physiological function of pyruvate-, α-ketoglutarate- and isocitrate dehydrogenases, and F₀F₁ ATPase are regulated by Ca²⁺. Consequently, rising Ca²⁺ in the mitochondrial matrix of stimulated cells could serve to stimulate Ca²⁺-sensitive dehydrogenases of the tricarboxylic acid (TCA) cycle, resulting in increased ATP synthesis as a result of the needs of a stimulated cell (McCormack et al. 1990). An increase in mitochondrial and cytosolic ATP, which depends on an increase in the mitochondrial Ca²⁺ concentration, was confirmed by direct measurement of ATP levels using a targeted luciferase probe (mtLUC) (Jouaville et al. 1999). Recent reports have indicated that inhibition of InsP₃R-dependent Ca²⁺ transfer from the ER to the mitochondria results in reduced ATP production, enhanced activation of AMP-activated protein kinase, and autophagy (Cardenas et al. 2010). Cells lacking all three InsP₃R isoforms are protected from nutrient deprivation and recover much faster than wild-type cells when nutrient supply is restored (Cardenas et al. 2010). Thus, by activating pro-survival autophagy, suppression of InsP₃R-mediated Ca²⁺ signaling represents a response mechanism to bioenergetic stress even in the presence of nutrients. Conversely, a constitutive low level of InsP₃R-mediated Ca²⁺ transfer to mitochondria under normal condition is required for autophagy suppression in the absence of specific agonist stimulation, and maintains optimal mitochondria bioenergetics by supporting oxidative phosphorylation (Cardenas et al. 2010).

The mitochondria is also considered a checkpoint in the intrinsic pathway of apoptosis, and Ca²⁺ plays a crucial sensitizing signal in the pro-apoptotic transition of the organelle, by establishing that the release of caspase cofactors, such as cytochrome c and a second mitochondria-derived activator of caspase/diablo, is the signal causing the assembly of the apoptosome and the commitment of the cell to apoptotic death (Szydlowska and Tymianski 2010). As to mechanisms of release, a key role is played by organelle fragmentation and swelling triggered by the opening of a large-conductance channel, the permeability transition pore (PTP) (Bernardi et al. 2006). Ca²⁺ is the most important trigger for PTP opening, acting in living cells in conjunction with a variety of pathological challenges. Otherwise, mitochondrial Ca²⁺ overload has been known to be a critical in the bioenergetics crisis related to cell death by necrosis (Szydlowska and Tymianski 2010). It is conceivable, that mitochondrial Ca²⁺ loading may play an important role, allowing a variety of toxic challenges to cause the release of caspase cofactors from the mitochondria resulting in the induction of cell death. Consequently, alteration of mitochondrial cellular response plays a role in the pathogenesis of human disorders.

Conclusions

Ca²⁺ loading from the ER and/or outside of the cell to the mitochondria controls pivotal metabolic processes in the mitochondria (Figure 1), and alterations in this movement of Ca²⁺ plays an important role in the pathologies of diverse human diseases. A decrease in cellular metabolism elicits various pathologies such as type-2 diabetes mellitus, insulin resistance, Alzheimer's disease, and cardiovascular disease. Alterations in cellular Ca²⁺ homeostasis, signs of ER stress, and decreases in mitochondrial membrane potential as well as reduced ATP levels are recurrent molecular events associated with these pathologies (Kelley et al. 2002). Additionally, modification of the contacts between the ER and mitochondria are also critical events in the regulation of cellular metabolism, and interruption of communication between the ER and mitochondria could underlie mitochondrial dysfunction and metabolic imbalance (Decuypere et al. 2011). There has been considerable progression in our understanding of the impact of mitochondrial Ca²⁺ signaling in the control of fundamental cell functions in terms of aerobic metabolism and the cell death pathway. The next challenges will be the identification of addition key transporters responsible for the transport of selective Ca²⁺ or other cations across mitochondrial membrane, of the relationship between transporter structure and function, and generation of transgenic animal models to allow understanding of the roles these transporters in the pathogenesis of human diseases.

Figure 1.  Metabolic communication between the endoplasmic reticulum (ER) and mitochondria via Ca^{2 +}. Ligand binds to growth factor receptor on the plasma membrane. The receptor generates an InsP₃ signaling molecule, which binds to the InsP₃R in the membrane of the ER. Ca^{2 +} in the ER is released through the InsP₃R to form high [Ca^{2 +}] microdomains located between the ER and mitochondria. Ca^{2 +} from the microdomains is translocated by VDAC in the outer membrane of the mitochondria. Subsequently, Ca^{2 +} is translocated by the MCU located in the inner membrane of mitochondria. Ca^{2 +} found in the matrix promotes the TCA cycle and ATP is generated by the electron transport cascade. Reduction of ATP production due to limited Ca^{2 +} efflux from the ER activates AMP-activated protein kinase (AMPK), which in turn stimulates autophagy by the cell. Thus, Ca^{2 +} in the mitochondria originates from the ER and regulates cellular metabolism.

Acknowledgements

Work in our laboratory is supported by the Canadian Institutes of Health Research (CIHR) and the Alberta Innovate-Health Solutions (AIHS).