The Involvement of Mg²⁺ in Regulation of Cellular and Mitochondrial Functions

Abstract

Mg²⁺ is an essential mineral with pleotropic impacts on cellular physiology and functions. It acts as a cofactor of several important enzymes, as a regulator of ion channels such as voltage-dependent Ca²⁺ channels and K⁺ channels and on Ca²⁺-binding proteins. In general, Mg²⁺ is considered as the main intracellular antagonist of Ca²⁺, which is an essential secondary messenger initiating or regulating a great number of cellular functions. This review examines the effects of Mg²⁺ on mitochondrial functions with a particular focus on energy metabolism, mitochondrial Ca²⁺ handling, and apoptosis.

1. Impact of Mg²⁺ on Cellular Functions and Intracellular Mg²⁺ Dynamics

Mg²⁺ is an essential mineral with pleotropic impacts on cellular physiology and functions [1, 2]. It acts as a cofactor of several important enzymes, especially those requiring ATP in order to be fully functional, such as the various protein kinases, proteins involved in nucleic acid metabolism, or ATPases involved in the transport of various ions [1, 2]. In addition, Mg²⁺ alters the electrophysiological properties of ion channels such as voltage-dependent Ca²⁺ channels and K⁺ channels [3]. The voltage-dependent block of N-methyl-D-aspartate receptor by Mg²⁺ [4, 5] represents an important phenomenon in the neurosciences. Finally, Mg²⁺ can affect the binding affinity of Ca²⁺ to specific Ca²⁺-binding proteins, such as calmodulin [6], S100 [7], troponin C [8], and parvalbumin [9, 10]. The effects of Mg²⁺ on Ca²⁺-handling proteins are responsible for the significant modification of intracellular Ca²⁺ dynamics and signalling [11]. In general, Mg²⁺ is considered as the main intracellular antagonist of Ca²⁺, which is an essential secondary messenger initiating or regulating a great number of cellular functions in various cells [12].

Recent progress in the field of Mg²⁺ transporter research has led to the identification of plasma membrane Mg²⁺ transporter SLC41A1 [13, 14], mitochondrial Mg²⁺ efflux system SLC41A3 [15], mitochondrial Mg²⁺ influx channel Mrs2 [16], and a mitochondrial Mg²⁺ exporter [17]. Substantial progress has also been achieved with respect to the regulation of whole body Mg²⁺ homeostasis [18]. These discoveries have shed new light on the importance of Mg²⁺ in cellular physiology including mitochondrial functions. Mitochondria have been demonstrated to be capable of both the accumulation of Mg²⁺ and the release of Mg²⁺ [19, 20]. Thus, mitochondria represent an important intracellular Mg²⁺ store. Significant amount of intracellular Mg²⁺ has also been shown to be localised within the lumen of the endoplasmic/sarcoplasmic reticulum (ER/SR) [21]. However, unlike mitochondria, the molecular mechanisms of Mg²⁺ transport through the ER membrane are not yet clear. Since impact of Mg²⁺ on cellular functions was summarised in recent reviews [1–3], we will deal, in this review, with the effects of Mg²⁺ on mitochondrial functions with a particular focus on energy metabolism, mitochondrial Ca²⁺ handling, and apoptosis (Figure 1).

Figure 1.

Regulation of mitochondrial functions by Mg²⁺. Mitochondrial Mg²⁺ activates (------>) three dehydrogenases in the mitochondrial matrix: pyruvate dehydrogenase (conversion of mitochondrial pyruvate to acetyl coenzyme A), isocitrate dehydrogenase (conversion of isocitrate to 2-oxoglutarate), and 2-oxoglutarate dehydrogenase (conversion of 2-oxoglutarate to succinyl coenzyme A). In addition, mitochondrial Mg²⁺ activates F₀/F₁-ATP synthase, which is the terminal complex of mitochondrial oxidative phosphorylation (OXPHOS). This regulatory activity contributes to mitochondrial energy metabolism. Mitochondrial Mg²⁺ inhibits (------|) Ca²⁺ transporters localised in the inner mitochondrial membrane: Ca²⁺-dependent permeability transition pore (PTP) opening that results in the release of a variety of compounds from mitochondria including Ca²⁺, mitochondrial Ca²⁺ uniporter (MCU), mitochondrial ryanodine receptor (RyR), and mitochondrial Na⁺/Ca²⁺ exchanger (NCX). This regulatory activity contributes to both intracellular and mitochondrial Ca²⁺ homeostasis. Cytoplasmic Mg²⁺ regulates mitochondrial Bax/Bak-dependent apoptosis, which is regulated by proteins of the Bcl-2 family such as Bcl-X_L, Bcl-2. TCA: tricarboxylic acid cycle/Krebs cycle, ACoA: acetyl coenzyme A, C: citrate, IC: isocitrate, OG: 2-oxoglutarate, SC: succinyl coenzyme A, S: succinate, F: fumarate, M: malate, OA: oxaloacetate, RaM: rapid mode of mitochondrial Ca²⁺ uptake, HCX: mitochondrial H⁺/Ca²⁺ exchanger, SLC41A3: mitochondrial Mg²⁺ efflux system, Mrs2: mitochondrial Mg²⁺ influx channel, VDAC: voltage dependent anion channel.

2. Impact of Mg²⁺ on Energy (Oxidative) Metabolism

The oxidation of coenzymes (reduced in glycolysis, reaction catalysed by pyruvate dehydrogenase complex, β oxidation, and Krebs cycle) in the mitochondrial respiratory chain and the consequent mitochondrial oxidative phosphorylation represent the major pathway of intracellular energy production in the form of ATP for all mammalian cells, except for erythrocytes. A small fraction of ATP is produced in the cytoplasm by the oxidation of glucose in the glycolysis pathway. Many of the glycolytic enzymes (hexokinase, phosphofructokinase, phosphoglycerate kinase, and pyruvate kinase) have previously been shown to be sensitive to Mg²⁺. The most important effect is attributable to the MgATP₂ complex, which is a cofactor for these enzymes, whereas other chelation forms are inactive or inhibitory [22].

The study of the impact of Mg²⁺ on the enzymes of energy metabolism in mitochondria began several decades ago [23, 24]. The earlier approach, which was focused on the description of the Mg²⁺ effect on isolated mitochondrial enzymes [25, 26], has subsequently been substituted by studies focused on the effect of Mg²⁺ on energy metabolism in isolated vital mitochondria [27–29] or vital cells [30, 31]. Some results obtained by the kinetic analysis of isolated enzymes have also been further analysed in more details by mathematical methods [32, 33]. Mg²⁺ has been documented to enhance the activity of three important mitochondrial dehydrogenases involved in energy metabolism. Whereas activities of isocitrate dehydrogenase (IDH) and 2-oxoglutarate dehydrogenase complex (OGDH) are stimulated directly by the Mg²⁺-isocitrate complex [25] and free Mg²⁺ [34], respectively, the activity of pyruvate dehydrogenase complex (PDH) is stimulated indirectly via the stimulatory effect of Mg²⁺ on pyruvate dehydrogenase phosphatase, which dephosphorylates and thus activates the pyruvate decarboxylase of PDH [35]. OGDH is the rate-limiting enzyme of the Krebs cycle and acts as an important mitochondrial redox sensor [36, 37]. The results obtained by the complex investigation of the impact of Mg²⁺ on ATP synthesis, the mitochondrial transmembrane potential, and respiration indicate that OGDH is the main step of oxidative phosphorylation modulated by Mg²⁺ when 2-oxoglutarate is the oxidisable substrate; with succinate, the ATP synthase is the Mg²⁺-sensitive step [29]. Indeed, Mg²⁺ has been shown to be the activator of ATP synthesis by mitochondrial F₀/F₁-ATPase [38, 39].

Taken together, the data suggest that Mg²⁺ has significant impact on the metabolic state, which is mediated by its stimulatory effect on the above-mentioned mitochondrial enzymes. However, the mitochondrial metabolic state seems, in turn, to affect the Mg²⁺ concentration of both the matrix [40] and the cytoplasm [41]. Finally, the effect of Mg²⁺ on energy metabolism partially interferes with the stimulatory effect of Ca²⁺ on energy metabolism and mitochondrial Ca²⁺ transport that are particularly important in excitable cells such as neurones [42, 43] and muscle cells [44]. Increase of extramitochondrial concentration of Mg²⁺ that was not associated with increase of Mg²⁺ concentration in mitochondrial matrix led in the presence of Ca²⁺ to the attenuation of state 3 respiration and stimulation of state 4 respiration [45]. This effect was attributed to the Mg²⁺-dependent inhibition of mitochondrial Ca²⁺ uptake (see further) that resulted in decrease of matrix Ca²⁺ concentration [45].

3. Involvement of Mg²⁺ in Regulation of Mitochondrial Ca²⁺ Transport

Mitochondria are important players in intracellular Ca²⁺ homeostasis and signalling [46, 47]. In response to specific signals, mitochondria are capable of both the active accumulation of intracellular Ca²⁺ and the release of Ca²⁺ from mitochondria via different Ca²⁺ transport mechanisms localised on mitochondrial membranes (Figure 1). Thus, they are considered as rapid-uptake slow-release buffers of cytosolic Ca²⁺ [48, 49]. In addition to cell signalling, mitochondrial Ca²⁺ plays an important role with respect to metabolism and cell survival [50, 51]. Several molecular mechanisms control mitochondrial Ca²⁺ transport [52].

The transport of Ca²⁺ through the outer mitochondrial membrane (OMM) is mediated via voltage-dependent anion channel (VDAC) that can be modulated in various ways [52], but little is known about the effect of Mg²⁺ on VDAC-dependent Ca²⁺ transport. An early study had shown that Mg²⁺ did not alter single channel activity but modified single current amplitudes in the lower conductance channel [53].

Active mitochondrial Ca²⁺ uptake is mediated by a specific transporter, namely the mitochondrial Ca²⁺ uniporter (MCU), which transfers Ca²⁺ through the inner mitochondrial membrane (IMM) at the expense of the proton gradient generated by the mitochondrial respiratory chain. The rate of uptake has been described to be proportional to the mitochondrial transmembrane potential [54], but, recently, the exponential dependence of the relative Ca²⁺ transport velocity on the mitochondrial transmembrane potential has received greater support [55, 56]. Another physiologically important question is associated with the low affinity of MCU for Ca²⁺ (apparent K_d 20–30 μM at 1 mM Mg²⁺) [57]. The discrepancy between the low Ca²⁺ affinity of the MCU observed in vitro and the high efficiency observed in vivo has been explained on the basis of the microheterogeneity of cytoplasmic Ca²⁺ rising during stimulation. The microdomains of high intracellular Ca²⁺ concentration (10–20 μM) have been suggested to be transiently formed in regions of close proximity to mitochondria and Ca²⁺ channels of the ER or of the plasma membrane [58]. MCU-mediated Ca²⁺ transport in isolated heart, kidney, and liver mitochondria is inhibited in the presence of 1.5 mM Mg²⁺ by approximately 50% in the heart and kidney and by 20% in the liver [59]. Similarly, the inwardly rectifying mitochondrial Ca²⁺ current displaying sensitivity to ruthenium red and selectivity to divalent cations, similar to that of MCU, is reduced by 0.5 mM of cytoplasmic Mg²⁺ concentration to 41% of its conductance in Mg²⁺-free solutions [60]. Moreover, mitochondrial Mg²⁺ loading has been shown to suppress MCU Ca²⁺-uptake rates [61]. The data of experimental studies were used for mathematical modelling of MCU-mediated Ca²⁺ transport suggesting a mixed-type inhibition mechanism for Mg²⁺ inhibition of the MCU function [62]. On the contrary, Mg²⁺ increased the rate of the active and ruthenium-red-sensitive accumulation of Ca²⁺ by isolated rat heart mitochondria [63]. The discrepancy has been attributed to the concentration of Ca²⁺ used for measurements. In the last-mentioned study [63], Ca²⁺ uptake was measured at 25 μM Ca²⁺, thus at a concentration that in the absence of Mg²⁺ is enough to open the permeability transition pore (PTP). Although the rate of Ca²⁺ transport mediated by MCU is inhibited by Mg²⁺, the net accumulation of Ca²⁺ in mitochondria was increased because of the Mg²⁺-mediated prevention of Ca²⁺ leakage from mitochondria via PTP.

Some controversial findings have been reported to be related to the mitochondrial accumulation of Ca²⁺ through IMM via the mitochondrial ryanodine receptor (mRyR). Western blot analysis, immunogold electron microscopy, and the high-affinity binding of [³H]-ryanodine indicate that a low level of mRyR is localised within IMM [64]. Similarly to MCU, mRyR is inhibited by low concentrations of ruthenium red (1–5 μM) and by Mg²⁺ [64]. However, the IMM localisation of RyRs by immunogold labelling has not been confirmed by another group [65]. Results obtained in our laboratory also argue against the significant physiological importance of mitochondrial Ca²⁺ uptake via mRyR, since only energised rat heart mitochondria are able to accumulate substantial amounts of Ca²⁺ and the accumulation is prevented by the submicromolar concentration of ruthenium red [63]. Finally, the group of Sheu [66] has suggested that, upon Ca²⁺ overload in the matrix, mRyR might be responsible for mitochondrial Ca²⁺ efflux, thus preventing the activation of PTP (see below).

Recent study documented that Mg²⁺ does not affect the rapid mode of mitochondrial Ca²⁺ uptake [67] that represents another mechanism of Ca²⁺ transport through the IMM distinct from MCU [68].

The main route of mitochondrial Ca²⁺ release has previously been demonstrated to depend on the Ca²⁺-induced release of Ca²⁺ from mitochondria (mCICR). The mechanism of mCICR involves the transitory opening of the PTP operating in a low conductance mode. Therefore, Ca²⁺ fluxes from mitochondria are a direct consequence of the mitochondrial depolarisation spike (mDPS) caused by PTP opening [69]. In vitro, both mDPS and mCICR can propagate from one mitochondrion to another, generating travelling depolarisation and Ca²⁺ waves. Mitochondria therefore appear to be excitable organelles capable of generating and conveying electrical and Ca²⁺ signals. In living cells, mDPS/mCICR is triggered by IP₃-induced Ca²⁺ mobilisation leading to amplification of the Ca²⁺ signals primarily emitted from the ER [69]. As documented in our laboratory, the opening of PTP in the low conductance mode depends significantly on the Mg²⁺ concentration [63]. This is in agreement with the previous study that documented the inhibitory effect of divalent cations including Mg²⁺ on Ca²⁺-dependent opening of PTP [70].

Two additional antiporters are suggested to play an important role with respect to mitochondrial Ca²⁺ release/efflux [51, 57]. In nonexcitable tissues (liver, kidney), such an antiport, appear to be predominantly an H⁺/Ca²⁺ exchanger, whereas in excitable tissues (heart, brain), it appears to be primarily a Na⁺/Ca²⁺ exchanger [71, 72]. The molecule responsible for the Na⁺/Ca²⁺ exchange was identified in 2010 [73]. A possible molecular candidate for the H⁺/Ca²⁺ exchange (Letm1) was reported in 2009 [74], although this proposal is still controversial [75, 76]. As suggested by Takeuchi and coworkers [51], further analysis is necessary to determine whether Letm1 is, indeed, the H⁺/Ca²⁺ exchanger mediating Ca²⁺ extrusion from mitochondria. The transport activity of the Na⁺/Ca²⁺ exchanger is inhibited by Mg²⁺ at concentration 2.5 mM [77], whereas Mg²⁺ does not inhibit the Ca²⁺ flux mediated by the H⁺/Ca²⁺ exchanger Letm1, even at ∼300-fold excess [75].

4. Mg²⁺ and Mitochondrial Apoptosis

Mitochondria play an important role in the process of the intrinsic pathway of apoptosis [78, 79]. They are both targets of proteins of the Bcl-2 family that are essential regulators of intrinsic apoptosis pathway initiation [79, 80], and the residence of proteins playing a crucial role in the execution of intrinsic apoptosis (cytochrome c, Smac/Diablo, apoptosis-inducing factor, and endonuclease G) [81]. In some cells, the extrinsic (receptor) pathway of apoptosis is connected to the intrinsic pathway via receptor-initiated cleavage of Bid protein, which is also a member of the Bcl-2 family, and the consequent translocation of truncated Bid (tBid) to the mitochondria [79, 81].

In contrast to the well-established role of Ca²⁺ in apoptosis [82], the role of Mg²⁺ has been largely ignored. Several in vitro studies have suggested the stimulatory role of Mg²⁺ in both the extrinsic and intrinsic pathways of apoptosis. Changes in cytosolic Mg²⁺ concentration have been observed in the glycodeoxycholate-induced apoptosis of hepatocytes [83], during the proanthocyanidin/doxorubicin-induced apoptosis in K562/DOX cells [84] and in the Fas ligand-induced apoptosis of B lymphocytes [85]. The elevation of intracellular Mg²⁺ observed in early phase of apoptosis has been explained by Mg²⁺ being necessary to stimulate the activity of Ca²⁺/Mg²⁺-dependent endonucleases, which are the executors of apoptosis. Patel et al. [83] have shown that the incubation of cells in Mg²⁺-free medium prevents the rise in intracellular Mg²⁺ and reduces nuclear DNA fragmentation. On the contrary, Chien and coworkers [85] have documented that an increase in cytosolic free Mg²⁺ is independent of the extracellular Mg²⁺ concentration and the source of the elevated intracellular Mg²⁺ has been suggested to be in the mitochondria. This suggestion is supported by the discovery of mitochondrial Mg²⁺ efflux and influx transporters [15, 16] and by experiments revealing the efflux of Mg²⁺ from mitochondria with preserved integrity (i. e., high transmembrane potential, no swelling) as the response to the apoptotic compound, gliotoxin [86]. Finally, the upregulation of Mrs2 has been shown to be responsible for the inhibition of the adriamycin-induced apoptosis of a gastric cancer cell line, probably by suppressing Bax-induced cytochrome c release from the mitochondria [87]. On the other hand, recent studies have documented both the elevation of mitochondrial [88] and the decrease of cytoplasmic [89] Mg²⁺ concentrations in some models of the induction of apoptosis.

Previous studies have also documented the impact of Mg²⁺ on cytochrome c release from mitochondria, an event that is followed by apoptosome formation and further progression of mitochondrial apoptosis [79]. Although a promoting effect of Mg²⁺ has been suggested, the impact of Mg²⁺ on cytochrome c release seems to depend on the mechanism of OMM permeability increase. The release of both Bax- [90] and tBid-induced cytochrome c [91] has been shown to be independent of the PTP pore but to be highly stimulated by Mg²⁺. On the contrary, Noxa-induced cytochrome c release is inhibited by Mg²⁺; this can be explained by the ability of Mg²⁺ to inhibit PTP [92], since PTP opening can result in the release of a variety of compounds from the mitochondria including that of cytochrome c leading to apoptosis [81].

5. Conclusions

Mitochondrial dysfunction has been implicated in the mechanisms of several serious human pathologies including metabolic [93, 94], cardiovascular [95], and neurodegenerative [96, 97] diseases. As we have discussed above, Mg²⁺ affects mitochondrial functions that have an important impact on cell survival. Recent work on Mrs2 knockdown HeLa cells has unambiguously revealed that the disruption of mitochondrial Mg²⁺ homeostasis has a dramatic impact on a cellular energy status and cell vulnerability [31]. Moreover, mitochondrial extruder SLC41A3 has been shown to be involved in the regulation of the whole-body Mg²⁺ balance [98]. These findings argue for more systematic research in the field of Mg²⁺ and mitochondria. Since mitochondria display significant cell and tissue heterogeneity [49, 99], the impact of mitochondrial Mg²⁺ on cellular physiology can also be anticipated to be cell- and tissue-type-dependent. Experiments on a variety of cell types will be important. In addition, the impact of Mg²⁺ on apoptosis initiation and execution in various cells has to be investigated in more detail. With respect to apoptosis, the cell-type specificity and the cause-consequence relations between apoptosis initiation and changes in the intracellular or mitochondrial concentration of Mg²⁺ are still unclear. Moreover, recent studies strongly point to the importance of ER-mitochondria interactions with respect to mitochondrial functions, Ca²⁺ homeostasis, and dynamics [100, 101]. Since the ER transport of Mg²⁺ is not as clear yet, the study of the transport of Mg²⁺ through the ER membrane and the possible impact of the luminal Mg²⁺ concentration on ER-mitochondria crosstalk and on mitochondrial Mg²⁺ transport and functions will be crucial. Finally, other processes are localised in the mitochondria, which are also considered as the main site of the intracellular production of reactive oxygen species. The effect of Mg²⁺ on these processes has not been discussed in this review, but some interest should be focused on this direction in the future.

Conflicts of Interest

The authors declare that there is no conflict of interest regarding the publication of this paper.