Mitochondrial Ca²⁺ channels: Great unknowns with important functions

Abstract

Mitochondria process local and global Ca²⁺ signals. Thereby the spatiotemporal patterns of mitochondrial Ca²⁺ signals determine whether the metabolism of these organelles is adjusted or cell death is executed. Mitochondrial Ca²⁺ channels of the inner mitochondrial membrane (IMM) actually implement mitochondrial uptake from cytosolic Ca²⁺ rises. Despite great efforts in the past, the identity of mitochondrial Ca²⁺ channels is still elusive. Numerous studies aimed to characterize mitochondrial Ca²⁺ uniport channels and provided a detailed profile of these great unknowns with important functions. This mini-review revisits previous research on the mechanisms of mitochondrial Ca²⁺ uptake and aligns them with most recent findings.

1. Introduction

Mitochondria have been recently recognized as multifunctional organelles that elementary impact on many different signaling pathways, thus, putting a new complexion on the long-known cellular power houses [1–4]. One distinguished feature of mitochondria that became evident by the utilization of newly developed mitochondria-targeted protein-based Ca²⁺ sensors [5,6] is the organelle’s active involvement during physiological Ca²⁺ signaling [7–10].

Herein, mitochondria were discovered to be far more than a passive Ca²⁺ sink that stores Ca²⁺ ions as Ca²⁺-(poly)_x-phosphate[11,12]. Indeed, mitochondrial Ca²⁺ handling turned out to represent a highly sophisticated mechanism that has multiple consequences for cells (Fig. 1A):

• First, mitochondria themselves constitute a Ca²⁺ target as these organelles house several Ca²⁺-sensitive proteins among which key metabolic enzymes, such as the dehydrogenases of the Krebs-cycle, translate Ca²⁺ elevation in the mitochondrial matrix to increased respiration and ATP production [13–17].

• Second, mitochondria crucially contribute to Ca²⁺ signaling by their ability to take up and release large amount of Ca²⁺ ions. By their potential to sequester cytosolic Ca²⁺, mitochondria are able to buffer Ca²⁺ in distinct region of the cell and keep spatial Ca²⁺ concentration low even under conditions of strong global Ca²⁺ mobilization upon cell stimulation, which significantly impacts on Ca²⁺-sensitive signal transduction within a cell [18–20]. Moreover, mitochondria are able to funnel Ca²⁺ to endoplasmic reticulum (ER) Ca²⁺ uptake sites [21,22] and accomplish ER Ca²⁺ replenishment during and after cell stimulation [22], an important process that ensures proper activity of the Ca²⁺-dependent ER chaperons of the protein folding machinery [23–26].

• Third, an excessive mitochondrial Ca²⁺ load sweeps cells to death by triggering either apoptosis or necrotic cell death [27–29], thus, signifying that mitochondrial Ca²⁺ uptake in any case of cellular Ca²⁺ mobilization essentially needs to be precisely regulated [27–29].

Fig. 1. Schematic illustration of mitochondrial Ca²⁺ uptake channels/carrier in the IMM, potential protagonists and the complexity if the mitochondrial environment.

(A) The complex environmental aspects of mitochondria in intact cells are illustrated. Local Ca²⁺ transfer from the ER via IP₃ mediated Ca²⁺ release. VDAC as porines in the OMM deliver Ca²⁺ ions to the MCU or Ca²⁺ exchanger at the IMM. (B) Electrophysiological characterizations revealed distinct mitochondrial Ca²⁺ channels: the MiCa in COS-7 cells and mCa1 as well as mCa2 in human ventricular myocytes. (C) Overexpression and siRNA mediated knock-down of UCP2/3 suggest a fundamental importance of these proteins for mitochondrial Ca²⁺ uniport. A genome-wide RNAi screen identified Letm1 as a mitochondrial Ca²⁺/H⁺ antiporter that significantly contributes to mitochondrial Ca²⁺ uptake in the physiological range of cytosolic Ca²⁺ elevation. (D) ER-mitochondria contact sites are stabilized by mitofusin 2 and probably other, so far unknown, proteins. The chaperon grp75 was shown to link the IP₃R to the VDAC in the OMM. Although proteins/factors that might link the VDAC to mitochondrial Ca²⁺ channels of the IMM have not been identified so far it is tempting to speculate that Ca²⁺ enters mitochondria via a Ca²⁺ tunnel spanning the OMM and IMM. (E) Evidence accumulated that kinases as well as Ca²⁺ and Ca²⁺-calmodulin differentially modulate the activities of mitochondrial Ca²⁺ channels.

Substantial studies over the last decades demonstrated that Ca²⁺ channels and Ca²⁺ exchanger in the inner mitochondrial membrane (IMM) establish Ca²⁺ transfer across the organelle’s inner membrane, while the ion transfer across the outer mitochondrial membrane (OMM) was thought to represent a rather uncontrolled process [30–33]. However, the latter view has been changed recently as regulatory mechanisms of the main Ca²⁺-permeable channels in the OMM, the voltage-dependent anion selective channels (VDAC1, 2 and 3), have been demonstrated [34–36] and their functional involvement in apoptosis was described [37–41]. While with the VDAC family the proteins for the Ca²⁺ transfer across the OMM are most likely identified, the proteins that are responsible for Ca²⁺ movements across the IMM are not completely identified so far (Fig. 1A). However, these mitochondrial Ca²⁺-shuttling proteins have been functionally well characterized although in many studies isolated mitochondria were used (reviewed in [42]) and the translations of such results to respective processes in intact cells, where mitochondria are embedded into an highly interactive environment, need cautiousness [43]. In this review we consider these aspects and intend to summarize recent progresses in the identification of functional and structural properties of mitochondrial Ca²⁺ channels of the IMM.

2. Characterization and identification of mitochondrial Ca²⁺ channels

Energized, respiring mitochondria are naturally destined to sequester Ca²⁺ due to their great negative membrane potential of the IMM (ψ_mito) that establishes a strong driving force for Ca²⁺ uptake into this organelle [9,44]. Moreover, mitochondrial are capable to store high amounts of Ca²⁺ in the mitochondrial matrix by the formation of Ca²⁺-(poly)_x-phosphates [12,31]. Mitochondrial Ca²⁺ uptake stimulates ATP production, but can also initiate cell death. Accordingly, the molecular mechanisms of mitochondrial Ca²⁺ uptake gained much attention during the last years.

2.1. Electrophysiological characterization of distinct mitochondrial Ca²⁺ channels

Recently, in two landmark publications that described the electrophysiological characterization of three highly selective Ca²⁺ channels in the IMM, which presumably account for the mitochondrial Ca²⁺ uniporter (MCU) phenomenon, were presented (Fig. 1B).

2.1.1. The MiCa

Applying the patch-clamp technique on mitoplasts (isolated mitochondria lacking their OMM) from COS-7 cells, the existence of a highly specific Ca²⁺ channel in the IMM has been convincingly demonstrated in 2004 by the laboratory of Clapham et al. [45]. This unique mitochondrial Ca²⁺ channel was referred to as MiCa. The electrophysiological characterization of MiCa showed that this channel is inwardly rectifying with a very high Ca²⁺ transport capacity making it very effective for Ca²⁺ uptake into energized mitochondria. These findings confirm visionary earlier studies in which the mitochondrial electrophoretic Ca²⁺ uniport was explored in isolated energized mitochondria [46,47] and reports that described the hexavalent cation ruthenium red (RR) and its related compound Ru360 as efficient inhibitors of the mitochondrial Ca²⁺ uniport in the nanomolar range [48]. Moreover, as expected for the MCU the permeability of MiCa to various divalent cations shows the following order: Ca²⁺ ≈ Sr²⁺ >> Mn²⁺ ≈ Ba²⁺, whereas MiCa is impermeable for Mg²⁺ ions. The monovalent ions K⁺ and Na⁺ do not contribute to the MiCa current in the presence of Ca²⁺, indicating the high Ca²⁺ selectivity of this mitochondrial Ca²⁺ channel. Notably, in this study 3–7 active channels per patch were found, which is a surprisingly high density of mitochondrial Ca²⁺ channels. Moreover, this study was of utmost significance as it was the first direct measurement of a mitochondrial Ca²⁺ channel in the IMM, thus, approving that mitochondrial Ca²⁺ uptake is indeed accomplished via a Ca²⁺ channel allowing the fast uniport of Ca²⁺ ions across the IMM (Fig. 1B).

2.1.2. The mCa1 and mCa2

In an outstanding work, two distinct mitochondrial Ca²⁺ channels have been recently electrophysiologically characterized in mitoplasts that were prepared from human ventricular myocytes [49]. These Ca²⁺-selective channels of the IMM are referred to as mCa1 and mCa2 and significantly differ in their single-channel amplitudes, opening times, open probabilities and their sensitivity to Ru360. Although the human mCa1 [49] share some characteristics such as its sensitivity to Ru360 with the MiCa [45] from COS-7 cells (an African Green Monkey SV40-transf’d kidney fibroblast cell line), the gating properties of both mitochondrial Ca²⁺ channels are considerable different, thus, possibly pointing to species and or tissue-specific heterogeneities among mitochondrial Ca²⁺ channels. Moreover, the coexistence of distinct mitochondrial Ca²⁺ currents, i.e. the Ru360-sensitive I_mCa1 and the rather Ru360-insensitive I_mCa2, in mitoplasts from the same origin (human ventricular myocytes) emphasize the need of different mitochondrial Ca²⁺ uptake pathways, which might be essential to properly integrate diverse cytosolic Ca²⁺ signals to mitochondrial Ca²⁺-induced metabolism in one given cell type. In this context the biophysical properties described for the mCa1 perfectly fit to the necessity of a mitochondrial Ca²⁺ channel facing the rapid and strong ER Ca²⁺ release sites. However, mCa2 with its lower single-channel amplitude, longer opening time and a higher open probability represents an ideal candidate to achieve efficient mitochondrial uptake of Ca²⁺ that rather slowly increases at mitochondrial Ca²⁺ uptake sites upon, e.g. Ca²⁺ entry via the store-operated Ca²⁺ entry (SOCE) pathway [2,50] (Fig. 1B). Notably, both mCa1 and mCa2 exhibit a high Ca²⁺ selectivity as it was reported for the MiCa. The relative divalent ion conductance of mCa1 and mCa2 for Sr²⁺, Mn²⁺, Ba²⁺ and Mg²⁺ has however not been tested so far.

2.1.3. Ca²⁺ carriers and exchangers

Besides the Ca²⁺-permeable channels there are data describing the existence of Ca²⁺ carriers and exchangers in the IMM that are thought to achieve mitochondrial Ca²⁺ efflux under physiological conditions [51]. Notably, the mitochondrial Ca²⁺/Na⁺ exchanger (NCX_mito) not only represent the main route for mitochondrial Ca²⁺ extrusion (for reviews see [2,51]) but may also contribute to mitochondrial Ca²⁺ uptake under certain conditions [43]. However, substantial novel findings regarding the physiological roles of the NCX_mito would require the molecular identification of this Ca²⁺-shuttling protein.

2.2. Molecular identification of mitochondrial Ca²⁺ channels

Already more than 30 years ago, attempts to isolate and purify Ca²⁺ channels from isolated mitochondria were initiated [52,53]. However, despite many efforts the mitochondrial Ca²⁺ channels of the IMM have not been explicitly identified on the molecular level so far. The clarification of the molecular identities of mitochondrial Ca²⁺ channels is a great challenge for the future and would be essential for the understanding of mitochondrial Ca²⁺ signaling as a fundamental physiological process that essentially contributes to various intracellular signaling pathways. Moreover, recent studies demonstrated the great participation of mitochondrial Ca²⁺ uptake to various pathological processes [1,10,54,55] in, e.g. neurodegeneration [56,57] and cardio-vascular diseases [58,59], thus, pointing to mitochondrial Ca²⁺ channels as attractive targets for the development of novel therapeutic strategies against different diseases [60] (Fig. 1C).

2.2.1. Glycoproteins as early potential candidates for mitochondrial Ca²⁺ channels

Early studies in the 1970s and 1980s that aimed to isolate and purify the mitochondrial Ca²⁺ uniporter suggested that glycoproteins might be elementary involved in the transfer of Ca² across the IMM [32,52,53,61]. Thereby, a almost 90% purified 18 and 75 kD fraction were isolated using affinity chromatography with labeled ¹⁰³Ru360 and Ca²⁺ uptake into phospholipids vesicles reconstituted with such preparations could be observed [62]. However, the purification and subsequent identification of mitochondrial Ca²⁺ channels has turned out to be difficult and has not been accomplished yet. Notably, in course of these attempts antisera against mitochondrial glycoprotein preparations were obtained that inhibited the Ca²⁺ uniport in isolated liver mitoplasts and reconstituted phospholipids vesicles (reviewed in [33]), thus, indicating the potential importance of these findings.

2.2.2. The novel uncoupling proteins (UCPs) 2 and 3 are fundamental for mitochondrial Ca²⁺ uniport

Uncoupling is a process that dissipates the proton gradient across the IMM of energized mitochondria whereby heat instead of ATP is generated [63]. The UCP1, also referred to as thermogenin, is a protein of the IMM that accomplishes uncoupling of respiration from ATP production, while the exact molecular mechanism by which UCP1 funnels protons from the inter membrane space into the mitochondrial matrix is still debated [64]. After the identification of UCP1 the so-called novel UCP2 and UCP3 were discovered [65–67]. Although these proteins share certain sequence homology with UCP1, their contribution to uncoupling and thermoregulation under physiological conditions could not be confirmed so far [68]. However, recently the impact of protein overexpression and siRNA-mediated knock-down of UCP2 and UCP3 on mitochondrial Ca²⁺ uptake in intact endothelial cells was tested in response to physiological Ca²⁺ mobilization [69]. Surprisingly, these experiments revealed that both proteins are fundamental for mitochondrial Ca²⁺ uniport as the capacity as well as the velocity of mitochondrial Ca²⁺ sequestration strictly correlated with the expression level of UCP2 and UCP3. Expression of UCP2/3 mutants confirmed the important role of these proteins for mitochondrial Ca²⁺ uptake and pointed to the predicted inter membrane loop 2 (IML2) to be elementary for the mitochondrial Ca²⁺ transport function of these proteins. Notably, in the IML2 domain, UCP2 and UCP3 share high sequence homology whereas this sequence considerable differs from that of UCP1. Very recently we continued experiments with UCP3 mutants and discovered two distinct sites in the IML2 that are essential for the mitochondrial Ca²⁺ transport function of UCP3. Interestingly one site in the IML2 of UCP3 emerged to be specifically required for mitochondrial uptake of intracellularly released Ca²⁺, while another distinct position was essential for mitochondrial sequestration of entering Ca²⁺ (manuscript submitted). The obvious importance of UCP2/3 for mitochondrial Ca²⁺ uniport was further validated by experiments using isolated liver mitochondria from UCP2^−/− mice [43,69]. In summary these data suggest that UCP2/3 are conductive subunits of a Ca²⁺-selective mitochondrial ion channel at the IMM, though further work is required to challenge this hypothesis (Fig. 1C).

2.2.3. Letm1 as mitochondrial Ca²⁺/H⁺ antiporter contributing to mitochondrial Ca²⁺ uptake

As a result of a very recent siRNA screening to identify mitochondrial Ca²⁺ shuttling proteins in Drosophila S2 cells, Letm1 was identified as a Ca²⁺/H⁺ antiporter of the IMM, while respective candidates for mitochondrial Ca²⁺ channels have not been described [70]. Letm1 was previously associated with the Wolf–Hirschhorn syndrome, a complex congenital syndrome that is caused by a monoallelic deletion of chromosome 4 [71]. Although Letm1 has been referred to as a mitochondrial protein with unclear function, initially Letm1 was characterized to contribute to electroneutral K⁺/H⁺ exchange in mitochondria thereby controlling the mitochondrial K⁺ homeostasis and volume [72]. At a first glance the findings that Letm1 particularly contributes to mitochondrial Ca²⁺ uptake at low cytosolic Ca²⁺ raises (<1 μM), while at higher Ca²⁺ concentration another uptake pathway, presumably the MCU got activated [70], is surprising as one would rather expect that a Ca²⁺/H⁺ antiporter preferentially exports Ca²⁺ from mitochondria (Fig. 1C). However, these findings are in line with other reports suggesting the existence of MCU-independent uptake pathways that are presumably accomplished by mitochondrial exchangers working in their reversed mode [43,50,73,74].

2.2.4. Assembly of protein complexes that establish mitochondrial Ca²⁺ channels

Little is known whether mitochondrial Ca²⁺ channels are protein complexes or not. However our recent finding that an expression of human UCP2/3 was ineffective to affect mitochondrial uptake in yeast [69] suggest that additional proteins are necessary to constitute Ca²⁺-permeable channels in the IMM. Thus, it is reasonable to speculate that additional proteins/factors are necessary to reassemble the Ca²⁺ transport function of UCP2 and UCP3 in artificial or heterologous systems. In line with these findings and in analogy to the so called mitochondrial transition pore (MTP), a large conductance pore that upon opening makes the mitochondrial membranes suddenly permeable for molecules with a molecular weight up to appr. 1.5 kDa [75,76], it seems feasible that the mitochondrial Ca²⁺ uniport channels also exhibit multiprotein complexes of IMM and OMM proteins (Fig. 1D).

Overexpression of the adenine nucleotide translocase (ANT), which is also known to be a component of the MTP [77], was shown to significantly reduce mitochondrial Ca²⁺ uptake in intact cells [78]. Although the overexpression of ANT might cause MTP opening and, thus, depolarization of the IMM, it is tempting to speculate that the reduced mitochondrial Ca²⁺ signals in ANT overexpressing cells are at least in part, the result of a disturbed composition of a presumable mitochondrial Ca²⁺ channel complex. The most prominent candidate of a protein of the OMM that probably physically interact with proteins of the IMM to constitute a mitochondrial Ca²⁺ channel spanning the IMM and OMM is VDAC [79]. Overexpression of VDAC in HeLa cells and skeletal myotubes enhanced mitochondrial Ca²⁺ uptake, indicating that this OMM porines are involved in the transfer of cytosolic Ca²⁺ into the lumen of mitochondria [80]. Notably, the chaperone glucose-regulated protein 75 (grp75) was found to link the inositol 1,4,5-trisphosphate receptor (IP₃R) to VDAC, which presumably enhances the transfer of Ca²⁺ from the ER towards mitochondria [81]. The exploration of the molecular basis of structural components of ER-mitochondria contact sites is currently a matter of intensive research [82,83]. Recently, mitofusin 2 was identified as a molecular component of such tethers that connect the ER with mitochondria, which was also elementary for mitochondrial Ca²⁺ uniport of Ca²⁺ that was mobilized from the ER [84]. The physical alliance between ER and mitochondria is also referred to as mitochondrial-associated ER membrane (MAM), which emerges to have important roles for Ca²⁺ signaling [83]. Interestingly, a recent study using electron tomography showed that in MAM the distance between ER and mitochondria is in the range of 10–25 nm, which would allow a direct interaction of proteins of the ER with proteins of the OMM [85]. Accordingly, it is reasonable that mitochondrial Ca²⁺ conducting proteins of the IMM might be assembled in a complex with MAM proteins, which substantially contribute to the gating of mitochondrial Ca²⁺ channels in intact cells (Fig. 1D).

3. Modulation of mitochondrial Ca²⁺ channels

3.1. Phosphorylation of mitochondrial Ca²⁺ channels

First conspicuous evidence that mitochondrial Ca²⁺ channels are targets of kinases came from the observation that an inhibition of the p38 mitogen-activated kinase (MAPK) with SB 202190 increased mitochondrial Ca²⁺ uptake in response to cell stimulation with an IP₃ generating agonist [86]. At a first glance this finding would indicate that mitochondrial Ca²⁺ channels exhibit serine/threonine-phosphorylation sites that, once phosphorylated negatively regulated the channel’s activity. Because other inhibitors of the p38MAPK failed to mimic the effect of SB 202190 [87], while structural related compounds without affecting this kinase activity, such as plant flavanoids, enhanced mitochondrial Ca²⁺ loading [88], the contribution of p38MAPK to the regulation of mitochondrial Ca²⁺ uniport was questioned and an alternative explanation suggesting that compounds such as SB 202190 directly bind to mitochondrial Ca²⁺ channels was discussed. However, the group of András Spät subsequently convincingly demonstrated that siRNA mediated knock-down of p38MAPK efficiently and specifically increased mitochondrial Ca²⁺ uptake upon cell stimulation with an IP₃ generating agonist [89]. Form this study and their subsequent intriguing work [90,91], the authors concluded that p38MAPK, novel isoforms of the protein kinase C (PKC) family and protein kinase D play central roles in the regulation of mitochondrial Ca²⁺ channels to prevent mitochondrial Ca²⁺ overload by explosive IP₃ mediated ER Ca²⁺ mobilization and hence protect cells from cell death. Further studies also point to different PKC isoforms that putatively modulate mitochondrial Ca²⁺ uptake channels: overexpression of PKCβ in HeLa cells was shown to reduce mitochondrial Ca²⁺ uptake, whereas overexpression of PKCζ increased mitochondrial Ca²⁺ transients upon cell stimulation with an IP₃ generating agonist [92] (Fig. 1E).

3.2. Regulation of mitochondrial Ca²⁺ channels by Ca²⁺

Early Ca²⁺ uptake studies with isolated mitochondria indicated that mitochondrial Ca²⁺ channels are activated by Ca²⁺ [93]. These experiments suggest a slow and allosteric activation of the mitochondrial Ca²⁺ uniport by Ca²⁺. In contrast, whole mitoplast patch-clamp experiments exclude a role for Ca²⁺ in activating mitochondrial Ca²⁺ channels [45], as a Na⁺ conductance in the absence of Ca²⁺ was recorded, indicating that Ca²⁺ is not essential for MiCa activity. The inconsistency of such datasets show that depending on how isolated mitochondria/mitoplasts have been prepared and depending on the overall experimental conditions an methods used to measure the functioning of mitochondrial Ca²⁺ channels, clearly different, even contradictory results can be obtained [94] vs. [43]. Moreover, the Ca²⁺ sensitivity of mitochondrial Ca²⁺ channels might be linked to signaling proteins such as calmodulin, which are fragilely or transiently associated with mitochondrial Ca²⁺ channels in intact cells. Accordingly, mitochondrial isolation might lead to a loss of these kind of interactions as such procedures are simply too invasive. This view is supported by findings that mitochondrial Ca²⁺ uniport is partially a Ca²⁺-calmodulin-gated process using permeabilized RBL-1 cells [95]. This study describes that mitochondrial Ca²⁺ uniport is activated via a Ca²⁺-calmodulin dependent mechanism, whereas cytosolic Ca²⁺ subsequently leads to an inactivation of mitochondrial Ca²⁺ channels, preventing further Ca²⁺ uptake by these organelles. The role of calmodulin in facilitating the activity of the mitochondrial Ca²⁺ uniport was further confirmed by Csordas and Hajnoczky [96]. However, a biphasic regulation of mitochondrial Ca²⁺ uptake channels points to complex mechanisms that tune the transit of Ca²⁺ across the IMM in order to avoid fatal mitochondrial Ca²⁺ overload during intracellular Ca²⁺ signaling. Moreover, Moreau and Parekh continued their intriguing studies and reported recently that the Ca²⁺-dependent inactivation of the mitochondrial Ca²⁺ uniporter is linked to proton-fluxes through the ATP-synthase [97], providing not only a mechanism of autoregulation of ATP synthesis but also a reasonable feedback mechanism between mitochondrial Ca²⁺ up-take and the organelle’s metabolic function.

In isolated liver and heart mitochondria a so-called rapid mode of mitochondrial Ca²⁺ upake (RaM) exists [98]. This process allows a fast pulsatile uptake of large amounts of Ca²⁺ but only for a short period of time, because RaM was shown to be quickly inactivated by Ca²⁺ via Ca²⁺ binding to an external site [99]. Notably, RaM is also sensitive to RR and Ru360, hence, it is possible that RaM is not a distinct mitochondrial Ca²⁺ channel but rather reflects a certain state of mitochondrial Ca²⁺ uniport channel(s) (Fig. 1E).

4. Conclusion

Recently promising progresses in the identification and characterization of mitochondrial Ca²⁺ transporters has been accomplished. Nevertheless, despite numerous studies convincingly characterize functional and structural aspects of mitochondrial Ca²⁺ uptake, our current picture on the actual proteins being involved remains rather vague. Intriguingly, the existence of various distinct mitochondrial Ca²⁺ channels that accomplish the transfer of Ca²⁺ across the IMM have been reported, and it is tempting to speculate that a species- and tissue-specific diversities of mitochondrial Ca²⁺ channels exists. Nevertheless, the experimental conditions and techniques used to characterize mitochondrial Ca²⁺ fluxes have to be taken into consideration if a general assertion is made. Thus, despite recent progresses, the identification of the molecular components of mitochondrial Ca²⁺ channels remains a challenging task in molecular physiology and awaits further intensive investigation.

Abbreviations

ANT

adenine nucleotide translocase

ER

endoplasmic reticulum

grp75

glucose-regulated protein 75

IML2

inter membrane loop 2

IMM

inner mitochondrial membrane

IP₃R

inositol 1,4,5-trisphosphate receptor

MAM

mitochondrial-associated ER membrane

MAPK

mitogen-activated kinase

mCa/MiCa

mitochondrial Ca²⁺ channel

MCU

mitochondrial Ca²⁺ uniporter

MTP

mitochondrial transition pore

NCX_mito

mitochondrial Ca²⁺/Na⁺ exchanger

OMM

outer mitochondrial membrane

PKC

protein kinase C

RaM

rapid mode of mitochondrial Ca²⁺ uptake

RR

ruthenium red

SOCE

store-operated Ca²⁺ entry

UCP

uncoupling protein

VDAC

voltage-dependent anion selective channels