The destiny of Ca2+ released by mitochondria
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Mitochondrial Ca2+ is known to regulate diverse cellular functions, for example energy production and cell death, by modulating mitochondrial dehydrogenases, inducing production of reactive oxygen species, and opening mitochondrial permeability transition pores. In addition to the action of Ca2+ within mitochondria, Ca2+ released from mitochondria is also important in a variety of cellular functions. In the last 5 years, the molecules responsible for mitochondrial Ca2+ dynamics have been identified: a mitochondrial Ca2+ uniporter (MCU), a mitochondrial Na+–Ca2+ exchanger (NCLX), and a candidate for a mitochondrial H+–Ca2+ exchanger (Letm1). In this review, we focus on the mitochondrial Ca2+ release system, and discuss its physiological and pathophysiological significance. Accumulating evidence suggests that the mitochondrial Ca2+ release system is not only crucial in maintaining mitochondrial Ca2+ homeostasis but also participates in the Ca2+ crosstalk between mitochondria and the plasma membrane and between mitochondria and the endoplasmic/sarcoplasmic reticulum.
KeywordsMitochondria Ca2+ dynamics NCLX Letm1 Cellular function
Mitochondria are crucial organelles in ATP production as well as in Ca2+ storage. They also serve as master switches determining cell fate on exposure to different stimuli [1, 2, 3]. Mechanisms of Ca2+ homeostasis in mitochondria have been extensively studied over the last half century, so the importance of mitochondrial Ca2+ in regulating mitochondrial functions is well recognized. Ca2+ enters mitochondria mainly via a mitochondrial Ca2+ uniporter, a protein known as MCU or CCDC109A [4, 5]. The characteristics and physiological and pathophysiological functions of this protein, and its associated proteins have been widely studied [6, 7, 8]. On the other hand, studies of the molecules responsible for Ca2+ release by mitochondria have just begun, although functional characterization of the release system started in the 1970s . The mitochondrial Ca2+ release system mainly consists of an Na+–Ca2+ exchanger and an H+–Ca2+ exchanger. The molecule responsible for the former (NCLX) was identified in 2010 . A possible molecular candidate for the latter (Letm1) was reported in 2009 , although this is still controversial.
It is now well understood that some mitochondria are in close contact with the plasma membrane and others with the endoplasmic reticulum (ER)/sarcoplasmic reticulum (SR). Although the molecular mechanisms of tethering of mitochondria to the plasma membrane are not well understood, several tethering protein complexes involved in interactions between mitochondria and the ER/SR have been identified, and details of the molecular mechanisms have been reviewed [12, 13]. It is believed that these interactions are important in modulating a variety of cellular functions.
In this paper we review recent progress in the study of mitochondrial Ca2+ release system, specifically, interactions between mitochondria and the ER/SR and interactions between mitochondria and the plasma membrane, and discuss their physiological and pathophysiological significance, focusing on the destiny of Ca2+ released by mitochondria.
Physiological roles of Ca2+ in mitochondria and released by mitochondria
Mitochondrial Ca2+ regulates diverse cellular functions
Ca2+ crosstalk between mitochondria and the plasma membrane and between mitochondria and the ER/SR
Ca2+ enters cells via Ca2+ channels in the plasma membrane, for example the voltage-gated Ca2+ channel (VDCC) and the Ca2+ release-activated Ca2+ channel (CRAC) or store-operated Ca2+ channel. The Ca2+ which enters the cell is efficiently taken up by mitochondria located near the plasma membrane [30, 31, 32, 33]. Lawrie et al.  found that after depletion of stored Ca2+, re-addition of extracellular Ca2+ evoked an increase in mitochondrial Ca2+ but not in cytoplasmic Ca2+ in human umbilical vein endothelial cell line ECV304, in which 14 % of mitochondria are located within 700 nm of the inner surface of the plasma membrane. They suggested that Ca2+ levels increase in microdomains beneath the plasma membrane, causing predominant Ca2+ uptake by mitochondria facing the microdomains. However, the contribution of the Ca2+ microdomains depends on cell type, because this phenomenon was not observed for a clone of a HeLa cell line, in which <6 % of mitochondria are located in the proximity of the plasma membrane. Park et al.  reported that store-operated Ca2+ influx via CRAC channels through the basolateral membrane led to predominant Ca2+ uptake by sub-plasmalemmal mitochondria in pancreatic acinar cells. These reports suggest Ca2+ communication, through plasma membrane Ca2+ channels, with mitochondria located in proximity to plasma membrane. Ca2+ communication in the opposite direction has also been suggested. Mitochondrial membrane depolarization suppressed store-operated Ca2+ entry in T lymphocytes and in rat basophilic leukaemia cells [34, 35]. Accordingly, bidirectional Ca2+ crosstalk between mitochondria and the plasma membrane is important in the regulation of cellular functions. As will be described below, the mitochondrial Na+-Ca2+ exchanger NCLX is involved in this Ca2+ crosstalk.
Taken together, Ca2+ crosstalk between mitochondria and the plasma membrane and between mitochondria and the ER/SR is important in controlling cellular functions. Understanding the roles of each molecule participating in the Ca2+ crosstalk is particularly important for fully understanding cellular physiological and pathophysiological functions.
Ca2+-transporting systems in mitochondria
Studies of mitochondrial Ca2+ dynamics started more than half a century ago. The existence of a respiration-dependent pathway of Ca2+ into isolated rat kidney mitochondria was reported in the early 1960s [49, 50]. In the 1970s, pathways of Ca2+ out of isolated rat heart mitochondria (Na+-dependent) and out of isolated rat liver mitochondria (Na+-independent and H+-dependent) were discovered [9, 51]. Since then, characteristics of mitochondrial Ca2+ dynamics have been extensively studied [1, 52, 53, 54].
The major pathways of Ca2+ uptake into and efflux out of mitochondria are summarized in Fig. 1. Ca2+ uptake into mitochondria is mainly mediated by a mitochondrial Ca2+ uniporter driven by a highly negative membrane potential (mitochondrial membrane potential ΔΨ is −150 to −180 mV) . Another uptake system is a rapid uptake mode (RaM) which might contribute to mitochondrial Ca2+ uptake from fast cytosolic Ca2+ transients. A mitochondrial ryanodine receptor is also reported to mediate Ca2+ uptake into rat heart mitochondria. Details are available in other reviews [1, 52]. It was not until the 2010s that the identities of the molecules responsible for the mitochondrial Ca2+ uniport were revealed. A regulator of the Ca2+ uniporter, MICU, was discovered in 2010 before cloning of the mitochondrial Ca2+ uniporter . MCU (or CCDC109A) was then discovered as a gene coding the mitochondrial Ca2+ uniporter [4, 5]. Very recently, characteristics of MCU knockout have been reported. Although MCU knockout in Trypanosoma brucei resulted in marked dysregulation of mitochondrial bioenergetics, causing autophagy and cell death , relatively minor alteration of basal energetics was observed for MCU knockout mice . MCU and its regulators have been reviewed in detail elsewhere [7, 8].
Ca2+ efflux from mitochondria is mainly mediated by two saturable pathways, an Na+-dependent (Na+–Ca2+ exchanger; benzodiazepines and CGP-37157-sensitive) pathway and an Na+-independent (H+–Ca2+ exchanger; ruthenium red-insensitive) pathway. Under pathophysiological conditions in which the PTP opens, PTP functions as a Ca2+ transporter from mitochondria. In the sections below we describe, in detail, the two physiological pathways of mitochondrial Ca2+ release.
Mitochondrial Na+–Ca2+ exchanger
Biophysical properties of the mitochondrial Na+–Ca2+ exchanger
The mitochondrial Na+–Ca2+ exchanger was first discovered by Carafoli et al.  in 1974 in isolated rat heart mitochondria. This Na+–Ca2+ exchange activity is found in a wide variety of tissues and is dominant in the heart, brain, skeletal muscle, parotid gland, adrenal cortex, and brown fat [52, 59]. The Na+–Ca2+ exchanger is also present in liver, kidney, and lung mitochondria, although its activity is weak . In tissues in which mitochondrial Na+–Ca2+ exchange activity is low, H+–Ca2+ exchange activity is of dominant importance in the release of Ca2+ from mitochondria .
One interesting characteristic of the mitochondrial Na+–Ca2+ exchanger, which is distinct from the plasmalemmal Na+–Ca2+ exchanger (NCX), is that Li+ can substitute for Na+ . This unique characteristic contributed to identification of NCLX, a gene responsible for the mitochondrial Na+–Ca2+ exchanger, as will be described in the next section. The stoichiometry (ion-exchange ratio) and the electrogenicity of the mitochondrial Na+–Ca2+ exchanger were controversial, but it was believed to be electroneutral [62, 63]. Our group clearly demonstrated, by use of permeabilized rat ventricular myocytes, that the mitochondrial Na+–Ca2+ exchanger is voltage-dependent and electrogenic, which suggests the stoichiometry is >3Na+ for one Ca2+ . We also predicted by computer simulation that the voltage dependence of the mitochondrial Na+–Ca2+ exchanger changes, the affinity becoming lower with mitochondrial membrane depolarization . Because of these features, the mitochondrial Na+–Ca2+ exchanger dynamically changes the exchange mode (forward or reverse) and modulates the mitochondrial Ca2+ concentration in a manner dependent on cytosolic Na+ concentration and mitochondrial membrane potential. Recently, several molecules which may modulate the mitochondrial Na+–Ca2+ exchanger have been identified. Gandhi et al.  reported that a deficiency of a PINK1, a 581-amino-acid protein consisting of a mitochondrial targeting motif and a serine/threonine kinase domain homologous with that of the Ca2+/calmodulin family, causes impairment of mitochondrial Ca2+ efflux via the mitochondrial Na+–Ca2+ exchanger. Mutations in the PINK1 gene are known to cause autosomal recessive Parkinson’s disease . Da Cruz et al.  showed that a stomatin-like protein 2 (SLP-2), a novel member of the stomatin superfamily found in several types of human tumour, negatively modulates mitochondrial Na+–Ca2+ exchange activity in HeLa cells, regulating the capacity of mitochondria to store Ca2+. Although detailed mechanisms underlying the regulation of mitochondrial Na+–Ca2+ exchange activity by these proteins have not yet been clarified, recent identification of NCLX as a mitochondrial Na+–Ca2+ exchanger will surely accelerate understanding of the mechanisms.
Cloning, tissue distribution, and cellular localization of NCLX
In 2004, two independent research groups reported the cloning of a new transporter mediating Na+–Ca2+ exchange, which was subsequently identified as a mitochondrial Na+–Ca2+ exchanger [66, 67]. Cai and Lytton employed a bioinformatics-based search of the GenBank™ database using a conserved amino acid sequence of the α-repeat regions of the K+-dependent Na+–Ca2+ exchanger (NCKX) gene family 2 (NCKX2) . The amino acid sequence of the identified clone was divergent from that of NCX and NCKX family members, but was slightly closer to that of NCKX. The clone was therefore named “NCKX6”. They also found an alternative spliced isoform of mouse NCKX6. Although the long isoform was retained in the ER fraction and was not functional when heterologously expressed in HEK293 cells, the short isoform was targeted in the plasma membrane and had K+-dependent Na+–Ca2+ exchange activity. Very soon after the publication by Cai and Lytton , Sekler’s group found the same clone during a search for the Na+–Zn2+ exchanger gene . In contrast with the report by Cai and Lytton , both long and short clones had K+-independent Na+–Ca2+ exchange activity and did not have Na+–Zn2+ exchange activity when heterologously expressed in HEK293 cells. They also found that Li+ can substitute for Na+ to release Ca2+ from the cells, so they named the clone “NCLX”. The discrepant results obtained by the two groups might be a result of different experimental conditions, i.e. Cai and Lytton  used Li+ as substituent for Na+ to examine K+-dependent Na+–Ca2+ exchange activity. This might have affected the responsiveness of the clone. In addition, FLAG epitope might have interfered with the sorting system in the Cai and Lytton  experiments. Anyway, both groups found that NCKX6/NCLX had Na+–Ca2+ exchange activity and broad tissue distribution, for example heart, pancreas, skeletal muscle, stomach, spleen, and brain.
Effects of NCLX reduction on cellular functions
Mitochondrial Ca2+ at rest
Mitochondrial Ca2+ transient
Cytosolic Ca2+ transient
Faster rise slower decline
Unchanged at steady state. Slightly accelerated during stimulation
Reduced insulin secretion
Faster rise slower decline
Smaller and slower
Reduced glutamate release
Impaired wound healing
Diminished cytosolic Ca2+ increase after BCR stimulation
Smaller and slower
Cycle length prolongation
NCLX in pancreatic β-cells (Fig. 4a)
NCLX in astrocytes (Fig. 4b)
Brain is another tissue highly expressing NCLX. Among the different kinds of cell constituting the brain, astrocytes constitute approximately half the volume. They express a large number of G protein-coupled receptors (GPCRs), linked to a diverse array of intracellular cascades including elevation of intracellular Ca2+. It is also well known that astrocytes release neurotransmitters called gliotransmitters, for example glutamate, ATP and d-serine, which bind to neuronal receptors to modulate synaptic transmission and activity [76, 77]. Thus astrocytes not only interact with neuronal activity but also modulate this activity via gliotransmitters. Although still controversial, several reports suggest that release of gliotransmitters by astrocytes depends on elevation of cytosolic Ca2+ [77, 78]. The increase of cytosolic Ca2+ in astrocytes occurs via the phospholipase C (PLC)/IP3 pathway. That is, upon GPCR activation, PLC hydrolyses the membrane lipid phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and IP3, activating the IP3 receptor (IP3R) and releasing Ca2+ from the ER. Parnis et al. , by use of a mouse astrocyte culture, examined how NCLX is involved in Ca2+ dynamics and the release of gliotransmitters. They used pericam-mito to sense mitochondrial Ca2+ and found that the resting mitochondrial Ca2+ level increased, and that the rise and decline of mitochondrial Ca2+ transients caused by extracellular ATP application became faster and slower, respectively, on knocking down NCLX. These results were very similar to those observed for pancreatic β-cells . Interestingly, NCLX knockdown reduced Ca2+ entry into the cytosol both from extracellular space and from the ER, but the effect on the former was stronger. Detailed analysis revealed that NCLX knockdown impairs store-operated Ca2+ entry (SOCE), indicative of strong Ca2+ crosstalk between mitochondria and the plasma membrane, as in pancreatic β-cells. NCLX knockdown also significantly reduced such processes as exocytotic glutamate release, in vitro wound closure, and proliferation, which may be regulated in a Ca2+-dependent manner .
NCLX in B lymphocytes (Fig. 4c)
NCLX in cardiomyocytes (Fig. 4d)
The heart is one of the most studied organs for investigation of the characteristics of the mitochondrial Na+–Ca2+ exchanger, which serves as a major Ca2+ release system [87, 88]. The roles of the mitochondrial Na+–Ca2+ exchanger in cardiac energetics have been widely investigated. For example, activation of the mitochondrial Na+–Ca2+ exchanger by increasing cytosolic Na+ causes a decrease of mitochondrial Ca2+, resulting in an imbalance in energy demand and supply or in an increase of ROS production [89, 90]. In contrast, the contribution of the mitochondrial Na+–Ca2+ exchanger to cytosolic Ca2+ transients and to action potential generation has been regarded as negligible, because the contribution of mitochondrial Ca2+ uptake to Ca2+ release from cardiomyocytes is as low as 1–2 % . However, we recently discovered that NCLX participates in modulation of action potential configuration and in regulation of the automaticity of HL-1, a spontaneously beating cardiac cell line originating from mouse atrial myocytes . The expression patterns of ion channels and transporters in HL-1 cells are similar to those in adult atrial myocytes, except that HL-1 cells highly express the T-type Ca2+ channel (I CaT) and the hyperpolarization-activated cation channel (I ha), which are known to be involved in the automaticity of cardiac pacemaker sinoatrial (SA) node cells [92, 93, 94, 95]. NCLX reduction using siRNA reduced NCLX protein expression by ~50 %, resulting in a ~50 % reduction of the rate of cytosolic Na+-dependent mitochondrial Ca2+ efflux, confirming that NCLX is responsible for mitochondrial Na+–Ca2+ exchange in HL-1 cells. Mitochondrial Ca2+ content, evaluated as the Ca2+ chelation-responsive fraction of the intensity of mitochondrial Ca2+ sensor pCase12-mito, was larger in NCLX knockdown cells. Although beat-to-beat change of mitochondrial Ca2+ was not observed in HL-1 cells, the result indicated that NCLX contributes to the steady state mitochondrial Ca2+ content in intact HL-1 cells. Cellular energetics seemed to be unaffected by NCLX knockdown, because there were no differences in cellular ATP content, mitochondrial membrane potential, or mitochondrial ROS between control cells and NCLX knockdown cells. This may be because protein expression of NCLX knockdown using siRNA was reduced by 50 % only.
An interesting finding was that NCLX knockdown caused marked prolongation of the cycle length of spontaneous action potentials and Ca2+ transients . Kinetic analysis of electrophysiological data and Ca2+ transients obtained by line scanning cells loaded with cytosolic Ca2+ indicator Fluo-4 revealed that NCLX knockdown slowed the upstrokes of both action potentials and cytosolic Ca2+ transients. SR Ca2+ dynamics, which is known to contribute to the upstroke of cytosolic Ca2+ transients, was evaluated by use of ER/SR Ca2+ FRET protein cameleon D1ER . As a result, SR Ca2+ content of NCLX knockdown cells was smaller and SR Ca2+ reuptake rate was slower (Fig. 5b), suggesting that the NCLX reduction-mediated prolongation of cycle length is related to compromised SR Ca2+ dynamics. The mechanisms underlying the NCLX reduction-mediated prolongation of cycle length was further studied with a newly constructed mathematical model of HL-1 cells . The HL-1 cell model well reproduces the spontaneous generation of action potentials and Ca2+ transients, and the prolongation of the cycle length induced by knocking down NCLX. The model analysis indicated that automaticity of HL-1 cells is determined by spontaneous Ca2+ leak from the SR. Simulation of NCLX reduction showed that Ca2+ supply from the mitochondria to the SR decreased to slow down the rate of spontaneous Ca2+ leak from the SR. The timing of Ca2+-induced Ca2+ release (CICR), activation of the inward current of the plasma membrane NCX (I NCX), and thus the timing of activation of voltage-dependent Na+ current (I Na) and voltage-dependent T and L type Ca2+ channels (I CaT and I CaL) was thus delayed, resulting in prolongation of the cycle length. Taken together, our combined experiments and simulations indicated that NCLX regulates the rhythmicity of HL-1 cells via crosstalk between mitochondria and SR Ca2+ dynamics. Considering that NCLX reduction resulted in modification of plasma membrane NCX activity, NCLX may also be indirectly involved in mitochondria–plasma membrane Ca2+ crosstalk in HL-1 cells. Interestingly, Opuni and Reeves reported the functional coupling of mitochondrial function, possibly mitochondrial Na+–Ca2+ exchange activity, and plasma membrane NCX activity in Chinese hamster ovary cells stably transfected with bovine cardiac NCX . Further analysis is necessary to elucidate the interaction of mitochondrial NCLX and plasma membrane NCX. Because HL-1 cells are derived from atrial myocytes, which have no automaticity under physiological conditions, NCLX may be involved in the abnormal automaticity of atria, for example atrial flutter or atrial ectopic tachycardia. It may also be possible that occurrence of arrhythmia in patients with mitochondrial disease [98, 99] is caused by abnormal NCLX function. Further analysis is required to clarify the involvement of NCLX in these arrhythmias.
Whether NCLX participates in the automaticity of normal pacemaker cells, sinoatrial (SA) node cells, is a big issue. Recently, Yaniv et al.  reported that mitochondrial Na+–Ca2+ exchange inhibitor CGP-37157 slowed the generation of automaticity of rabbit SA node cells, suggesting that mitochondrial Na+–Ca2+ exchange is involved in generation of the automaticity of SA node cells. However, because CGP-37157 also blocks I CaL, which is related to generation of the automaticity of SA node cells, a nonspecific effect of CGP-37157 on I CaL cannot be ignored. In addition, the automaticity of the SA node cells they used significantly depends on spontaneous and local subsarcolemmal Ca2+ releases from SR, the so called “Ca2+ clock” mechanism . However, this automaticity mechanism has been controversial. It has been accepted for a long time that I ha and a variety of other inward membrane currents determine the automaticity [102, 103, 104]. This mechanism is called the “membrane clock”. Whether NCLX contributes to the automaticity of all types of SA node cells, including the cells driven by “membrane clock”, must be studied.
We examined the contribution of NCLX using mathematical models of SA node cells . In the original SA node model developed by Yaniv et al. , complete reduction of mitochondrial Na+–Ca2+ exchange resulted in prolongation of cycle length by 2.7 % only. The larger effect reported by Yaniv et al.  is probably because of their simultaneous reduction of the amplitude factor of SERCA. To further test the contribution of NCLX to generation of the automaticity of SA node cell models, we incorporated our model of mitochondrial Ca2+ dynamics into two representative SA node cell models: a membrane clock model by Himeno et al. [102, 103] and a Ca2+ clock model by Maltsev and Lakatta . In both models, NCLX reduction reduced the SR Ca2+ content, supporting the idea of Ca2+ communication between mitochondria and the SR in SA node cells. However, the effect on the automaticity was different between the models. The cycle length was prolonged in the Maltsev and Lakatta model whereas it was shortened in the Himeno model . Furthermore, model analysis revealed that cytosolic Na+ and Na+-permeable inward current (sustained inward current I st)  are crucial factors distinguishing the effect of NCLX on pace-making in the two models. In the Himeno model, NCLX reduction reduced cytosolic Ca2+, which decreased the inward I NCX thus reducing the cytosolic Na+ concentration. This increased the amplitude of the inward I st and overcame the decrease of inward I NCX, accelerating the firing rate. In contrast, the amplitude of I st is set smaller in the Maltsev and Lakatta model so that NCLX reduction only reduces inward I NCX and slows diastolic depolarization, decelerating the firing rate . Accordingly, it is likely that the Ca2+ communication between mitochondria and the SR via NCLX functions also in SA node cells. However, it is necessary to investigate quantitatively how much the ‘‘Ca2+ clock’’ mechanism, cytoplasmic Na+, and I st contribute to SA node automaticity.
Mitochondrial H+–Ca2+ exchanger
H+–Ca2+ exchange has a dominant effect on release of Ca2+ from mitochondria in tissues in which mitochondrial Na+–Ca2+ exchange activity is low, for example the liver, kidney, lung, and smooth muscle . The H+–Ca2+ exchanger also occurs in the heart, though the activity is weak [61, 107].
The stoichiometry of the H+–Ca2+ exchanger is regarded as 2H+ for 1Ca2+, being electroneutral. However, because the rate of efflux via the H+–Ca2+ exchanger decreases with increasing pH gradient in rat isolated liver mitochondria, it is suggested that the mechanism is not a passive Ca2+ for 2H+ exchanger, but an active Ca2+ for 2H+ exchanger .
The molecular identity of the mitochondrial H+–Ca2+ exchanger is still controversial. The candidate is the Letm1 (leucine–zipper–EF hand-containing transmembrane region). Jiang et al.  conducted high-throughput RNA interference screening of Drosophila genes and identified a gene affecting mitochondrial Ca2+ and H+, the human homolog of which is Letm1. By using digitonin-permeabilized S2 or 293 cells expressing mitochondrial Ca2+ sensor protein pericam, or by using purified Letm1 reconstituted in liposomes, they found that Letm1 mediates H+–Ca2+ exchange. However, because a drastic reduction of mitochondrial Ca2+ uptake was observed when Letm1 protein expression was suppressed, and because H+–Ca2+ exchange via Letm1 was sensitive to an inhibitor of mitochondrial Ca2+ influx, ruthenium red, Letm1 is regarded as mediating Ca2+ influx into mitochondria, at least at low cytosolic Ca2+ level. This idea was confirmed by subsequent work by Jiang et al.  in which Letm1 knockdown in 293 cells resulted in dramatically reduced mitochondrial Ca2+ content. In the same work they produced Letm1 knockout mice and found that Letm1 homozygous knockout mice were embryonic lethal and so were half of the heterozygous knockout mice. The surviving mice had altered glucose metabolism, impaired control of brain ATP levels, and increased seizure activity, suggesting involvement of Letm1 in the pathology of Wolf–Hirschhorn syndrome, in which Letm1 is known to be one of the genes deleted . Nowikovsky et al.  analysed theoretically the direction of Ca2+ flux through the mitochondrial membrane in energized mitochondria with different H+:Ca2+ stoichiometry. They proposed that Ca2+ enters mitochondria with 1H+:1Ca2+ whereas Ca2+ leaves mitochondria with 2H+:1Ca2+ or 3H+:1Ca2+ stoichiometry under physiological respiration conditions. Tsai et al.  reported that Letm1 mediates the electroneutral 2H+:1Ca2+ antiport and is insensitive to ruthenium red, by using Letm1 reconstituted proteoliposome. These results combined with the theoretical analysis by Nowikovsky et al.  strongly suggest that Letm1 is the long-awaited molecular identity of the mitochondrial H+–Ca2+ exchanger. There is, currently, no reasonable explanation of the discrepancy of the sensitivity of Letm1 to ruthenium red. We independently found that a substantial part of the Ca2+ efflux from mitochondria was independent of cytosolic Na+ when mitochondria were loaded with a lower concentration of Ca2+ than when examining Na+–Ca2+ exchange activity using A20 B lymphocytes . This fraction, which probably represents the H+–Ca2+ exchange system, was sensitive to ruthenium red. Further analysis is necessary to determine whether Letm1 is, indeed, the H+–Ca2+ exchanger mediating Ca2+ extrusion from mitochondria.
Ca2+, not only inside mitochondria but also released from mitochondria, is crucially important in regulating a variety of cellular physiological functions. Identification of the molecules responsible for the pathways of Ca2+ release enables us to isolate the contributions of these molecules. For example, NCLX has been shown to participate in insulin secretion in pancreatic β-cells, glutamate release in astrocytes, Ca2+ responsiveness to BCR stimulation in B lymphocytes, and generation of the spontaneous rhythmicity of cadiomyocytes, via Ca2+ crosstalk between mitochondria and the plasma membrane and/or between mitochondria and the ER/SR (Fig. 4). However, many issues remain unanswered. One is the question of whether mitochondrial Ca2+ release proteins are indeed located in the tethering spots between mitochondria and the ER/SR or between mitochondria and the plasma membrane. Another is the contribution of the mitochondrial Ca2+ release system to mitochondrial energetics. Distinct phenotypes of NCLX knockdown cells related to mitochondrial energetics in cardiomyocytes and in pancreatic β-cells have not been observed, while Letm1 knockout mice had impaired mitochondrial energetics. Complete knockout of NCLX in mice or cells will clarify the matter.
This work was supported by JSPS Kakenhi grant numbers 25136707, 23689011, and 23390042 (A.T. and S.M.).
Conflict of interest
The authors declare that they have no conflict of interest.
- 5.Baughman JM, Perocchi F, Girgis HS, Plovanich M, Belcher-Timme CA, Sancak Y, Bao XR, Strittmatter L, Goldberger O, Bogorad RL, Koteliansky V, Mootha VK (2011) Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter. Nature 476:341–345PubMedCentralPubMedGoogle Scholar
- 13.van Vliet AR, Verfaillie T, Agostinis P (2014) New functions of mitochondria associated membranes in cellular signaling. Biochim Biophys Acta (in press). doi: 10.1016/j.bbamcr.2014.03.009
- 58.Pan X, Liu J, Nguyen T, Liu C, Sun J, Teng Y, Fergusson MM, Rovira II, Allen M, Springer DA, Aponte AM, Gucek M, Balaban RS, Murphy E, Finkel T (2013) The physiological role of mitochondrial calcium revealed by mice lacking the mitochondrial calcium uniporter. Nat Cell Biol 15:1464–1472PubMedGoogle Scholar
- 60.Haworth RA, Hunter DR, Berkoff HA (1980) Na+ releases Ca2+ from liver, kidney and lung mitochondria. FEBS Lett 110:210–218Google Scholar
- 75.Proverbio MC, Mangano E, Gessi A, Bordoni R, Spinelli R, Asselta R, Valin PS, Di Candia S, Zamproni I, Diceglie C, Mora S, Caruso-Nicoletti M, Salvatoni A, De Bellis G, Battaglia C (2013) Whole genome SNP genotyping and exome sequencing reveal novel genetic variants and putative causative genes in congenital hyperinsulinism. PLoS ONE 8:e68740PubMedCentralPubMedGoogle Scholar
- 86.Kim B, Takeuchi A, Matsuoka S (2013) Mitochondrial NCX controls directional migration of B lymphocyte. J Physiol Sci 63:S136Google Scholar
- 105.Takeuchi A, Matsuoka S (2014) Mitochondrial Na–Ca exchanger NCLX-mediated mitochondria-sarcoplasmic reticulum Ca crosstalk and cardiomyocyte automaticity. J Physiol Sci 64:S68Google Scholar
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