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Ca2+ exchanger, NCLX
Seminars in Cell and Developmental Biology 94 (2019) 59–65
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Review
Functional properties and mode of regulation of the mitochondrial Na+/ Ca2+ exchanger, NCLX Marko Kostic, Israel Sekler
T
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Department of Physiology and Cell Biology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel
A R T I C LE I N FO
A B S T R A C T
Keywords: NCLX MCU Mitochondrial Ca2+ signaling Ca2+ and neurodegeneration
Mitochondrial Ca2+ transient is the earliest discovered organellar Ca2+ signaling pathway. It consist of a Ca2+ influx, mediated by mitochondrial Ca2+ uniporter (MCU), and mitochondrial Ca2+ efflux mediated by a Na+/ Ca2+ exchanger (NCLX). Mitochondrial Ca2+ signaling machinery plays a fundamental role in linking metabolic activity to cellular Ca2+ signaling, and in controlling local Ca2+ concertation in distinct cellular compartments. Impaired balance between mitochondrial Ca2+ influx and efflux leads to mitochondrial Ca2+ overload, an early and key event in ischemic or neurodegenerative syndromes. Molecular identification of NCLX and MCU happened only recently. Surprisingly, MCU knockout yielded a relatively mild phenotype while conditional knockout of NCLX led to a rapid fatal heart failure. Here we will focus on recent functional and molecular studies on NCLX structure and its mode of regulation. We will describe the unique crosstalk of this exchanger with Na+ and Ca2+ signaling pathways in the cell membrane and the endoplasmic reticulum, and with protein kinases that posttranslationally modulate NCLX activity. We will critically compare selectivity of pharmacological blockers versus molecular control of NCLX expression and activity. Finally we will discuss why this exchanger is essential for survival and can serve as an attractive therapeutic target.
1. Introduction Cellular Ca2+ signaling is of central importance for numerous cellular functions, echoing the famous saying of the late Nobel laureate Otto Loewi: "Ja, Kalzium, das ist alles" (Yes, calcium is everything). Mitochondria were among the earliest cellular organelles investigated for Ca2+ signaling. Early studies conducted in isolated mitochondria during 1960s showed that these organelles are capable of taking up large amounts of Ca2+ [1,2]. These early studies also indicated that the mitochondrial Ca2+ uptake is powered by, and strongly dependent on, the steep mitochondrial membrane potential (ΔΨm), as mitochondrial depolarization by protonophores or uncouplers abolished mitochondrial Ca2+ uptake. Subsequent studies have found that following the influx of Ca2+ into the mitochondria, Ca2+ could be pumped out of mitochondria primarily by a Na+-dependent process [3]. The focus and interest in mitochondrial Ca2+ signaling however waned with the development of the first fluorescent probes for cytosolic Ca2+. Analysis of Ca2+ changes using these probes suggested that cytosolic Ca2+ spikes in most non-excitatory cell types are reaching concentrations of hundreds of nM at most. These values were much smaller than the apparent affinity for mitochondrial Ca2+ uptake (tens of μM Ca2+ concentration) that was previously determined in isolated ⁎
mitochondria, thus indicating that mitochondrial Ca2+ uptake is of no physiological significance. These sober conclusions, taken together with the emergence of the endoplasmic reticulum (ER) as a major Ca2+storing organelle in the cell, led to a shift of interest from mitochondria to ER. For about 20 years, the mitochondrial Ca2+ signaling was largely abandoned. The change came in the 1990s by key experiments performed by Rizzuto and Pozzan. They were the first to use geneticallyencoded Ca2+ probes-the aequorins that were targeted to the mitochondrial matrix. In contrast to the results obtained in isolated mitochondria, many of the Ca2+ signals triggered in the ER and cytosol in cells were effectively propagated into the mitochondria [4,5]. These landmark experiments revived the discipline of mitochondrial Ca2+ signaling. The discrepancy between the in vitro and cellular results was resolved by subsequent demonstration that Ca2+ rise within the cell is not homogenously distributed, but is order of magnitudes higher in the so-called “hot spots”-cellular domains at mitochondria found in the vicinity of the plasma membrane or at ER-junctions [6]. Subsequent studies confirmed the role of specialized interaction junction between the mitochondria and ER where the flow of Ca2+ is particularly efficient and intense [7]. The second landmark breakthrough that occurred twenty years later were the molecular discoveries of the mitochondrial Na+/ Ca2+ exchanger, NCLX [8], and shortly afterwards of the
Corresponding author. E-mail address: [email protected] (I. Sekler).
https://doi.org/10.1016/j.semcdb.2019.01.009 Received 11 November 2018; Received in revised form 14 January 2019; Accepted 14 January 2019 Available online 30 January 2019 1084-9521/ © 2019 Elsevier Ltd. All rights reserved.
Seminars in Cell and Developmental Biology 94 (2019) 59–65
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mitochondrial Ca2+ uniporter-MCU [9,10]. The earlier and recent studies have identified several key roles for mitochondrial Ca2+ signaling: 1) Ca2+ rise is upregulating the activity of at least three enzymes in the Krebs cycle and that of the Fo/F1 ATPase [11–13]. Therefore, mitochondrial Ca2+ transient is an essential link between cytosolic Ca2+ signaling and changes in energy demand required for the activation of numerous downstream kinases and motor proteins; 2) In addition, the mitochondrial network is not static but is moving toward cell regions where Ca2+ signaling is particularly intense, predominantly at the vicinity of the ER and plasma membrane, and regulates the local Ca2+ concentrations at these domains [14]. Because major Ca2+ channels at the cell membrane and ER are allosterically regulated by cytosolic Ca2+, the control of cytosolic Ca2+ by the mitochondria strongly regulates activity of these channels. Most notable examples are the L-type Ca2+ channel (LTCC) [15], the ryanodine receptor (RyR) [16], transient receptor potential cation channel subfamily V member 1 (TRPV1) [17] or the store-operated Ca2+ channel (SOC) [18]; 3) In major pathophysiological syndromes, such as brain and cardiac ischemia or in many neurodegenerative syndromes, mitochondrial Ca2+ transient is impaired, often leading to the toxic accumulation of mitochondrial free Ca2+, termed mitochondrial Ca2+ overload [19–23]. Mitochondrial Ca2+ overload is induced by imbalance between mitochondrial Ca2+ uptake and removal, and often leads to mitochondrial swelling, opening of the mitochondrial permeability transition pore and initiation of apoptotic or necrotic cell death [24,25]. In this review we will focus on the mitochondrial Ca2+ removal, in particular on NCLX.
of the Na+/Ca2+ superfamily that diverged early on from the other two families, the NCX and NCKX. Surprisingly, in contrast to all other known members of the superfamily, it conducted an active Li+/Ca2+ exchange as well. Based on this unique Li+-dependent Ca2+ transport we termed it Na+/Li+/Ca2+ exchanger (NCLX) and reasoned that it may be linked to the long sought mitochondrial Na+/Ca2+ exchanger [32]. Western blot analysis revealed that NCLX was found in the mitochondrial fraction, and immunogold electron microscopy (EM) analysis showed that NCLX is found in the inner mitochondrial membrane. Functional analysis of mitochondrial Ca2+ transport showed that overexpression of NCLX enhanced, while silencing of NCLX impaired mitochondrial Ca2+ efflux [8]. Moreover, Li+ and Na+ fully supported mitochondrial Ca2+ efflux mediated by NCLX. All these results converged upon conclusion that NCLX is the long sought mitochondrial Na+/Ca2+ exchanger. 3. Catalytic site of NCLX The 3D structure of the archaebacterial Na+/Ca2+ exchanger NCX_Mj and the homologous H+/Ca2+ exchanger, CAX_Af provided an important handle to study NCLX transport sites [33–35]. A 3D model of NCLX using ROBETTA server indicated that despite the modest sequence homology of NCLX with its archaebacterial homologues they share a strikingly similar arrangement of the Na+ and Ca2+ binding sites [36]. The unique ability of NCLX to conduct both Na+/Ca2+ and Li+/Ca2+ transport makes it a good system to analyze monovalent cation selectivity. Mutagenesis screening showed that mutations in the α1 and α2 catalytic domains confer Li+ over Na+ selectivity. However, only mutation in a single residue of the α2 catalytic domain (D471) conferred Na+ over Li+ selectivity [36]. Another study that modeled NCLX according to NCX_Mj found that despite the strong 3D similarities of the Na+ and Ca2+ binding sites, nine coordinating residues of NCLX are distinct [37]. Replacing these residues on the backbone of Na+ selective NCX_Mj was followed by NCLX-like Li+-dependent Ca2+ exchange of the NCX Mj, thus indicating that this set of residues is required to confer the unique monovalent cation selectivity of NCLX. Future 3D structures of NCLX based on cryo EM or crystal structure will be required to determine precise NCLX ion transport site. In addition, it is important to note that NCX_Mj and other bacterial homologues lack the regulatory domain that apparently plays a key role in controlling the exchanger activity. The 3D structures of the regulatory domains of both NCX and NCLX are therefore required to understand the structural basis of their mode of regulation of ion transport and how they interact with the transport site.
2. Functional and molecular discovery of the mitochondrial Na+/ Ca2+ exchanger Activity of the mitochondrial Na+/Ca2+ exchanger was first identified in isolated cardiac mitochondria that were loaded with Ca2+ [3]. In the absence of Na+, no Ca2+ efflux was observed. However, when Na+ was added, it triggered a rapid mitochondrial Ca2+ efflux. A remarkable feature of the mitochondrial exchanger was that Li+ could replace Na+ in catalyzing mitochondrial Ca2+ efflux. This unique functional trait was vital for later molecular identification of the mitochondrial Na+/Ca2+ exchanger [26]. Subsequent studies found that mitochondrial Na+/Ca2+ exchange is a ubiquitous pathway found in many cell types and critical for many physiological activities. One notable example was found in brown adipose tissue (BAT) during norepinephrine-triggered uncoupled respiration, where Ca2+ that accumulated in BAT mitochondria could be subsequently removed only in the presence of Na+ [27]. Moreover, inhibition of mitochondrial Ca2+ efflux in the absence of Na+ severely impaired uncoupled respiration, underscoring the vital role of the exchanger in controlling the metabolic activity in BAT. Other studies have emphasized the unique divalent cation selectivity of NCLX for Ca2+ over Mg2+, Mn2+ and Ba2+ [3,28]. The use of matrix targeted Na+ dyes, most notably CoroNa Red, confirmed that the Ca2+ efflux mediated by the mitochondrial Na+/Ca2+ exchanger is coupled to mitochondrial Na+ influx. Effort to identify the mitochondrial Na+/Ca2+ exchanger gene started already in 1990s. Polypeptide candidates of ˜60 kDa and ˜110 kDa molecular weights were purified from beef heart [29,30]. Proteoliposmes reconstituted with these exchanger candidates showed Na+- and Li+-dependent Ca2+ efflux. Furthermore, antibody raised against the polypeptides and blockers of this exchanger inhibited Na+and Li+-dependent Ca2+ efflux. Moreover, the exchanger was highly sensitive to degradation by mitochondrial Ca2+-dependent protease μCalpain [31]. The molecular identification of NCLX as the mitochondrial Na+/ 2+ Ca exchanger started when we cloned and functionally analyzed a new member of the Na+/Ca2+ exchanger superfamily. A phylogenic analysis indicated that this gene is a single member of a distinct family
4. Na+-dependent functions of the mitochondrial Na+/Ca2+ exchanger Most of the interest and focus regarding the mitochondrial Na+/ Ca exchanger is about its Ca2+ signaling role. An often-overlooked feature of this transporter is that it transports 3Na+ per 1Ca2+, and is therefore transporting more Na+ ions in than Ca2+ ions out of the mitochondria. The knowledge and interest on the role of Na+ in cell signaling is much more limited than that of Ca2+. The general notion was that changes in transmembrane Na+ gradient are small, largely insignificant and that the major role of Na+ is in controlling the electrical activity in excitable cells. In addition, the quality of cellular fluorescent Na+ reporters is far lower than that of pH- or Ca2+- fluorescent reporters, thus limiting our ability to monitor changes in the concertation of Na+ [38]. Nevertheless, several key and early studies underscored the role of mitochondrial Na+ signaling by the mitochondrial Na+/Ca2+ exchanger. First, analysis of affinity of the exchanger to Na+ showed that it is ˜7 mM, close to the resting level of cytosolic Na+ [30,39]. Thus, even a small change in cytosolic Na+ will have a very strong impact on the rate of the exchanger activity (Fig. 1). Indeed, studies in astrocytes 2+
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Fig. 1. Mode of regulation and physiological/pathophysiological roles of NCLX. Calcium enters the cell by multiple pathways at the plasma membrane (PM), such as store-operated Ca2+ channel (SOC), L-type voltagegated Ca2+ channel (LTCC) or cationic channel TRPV1. Cytosolic Ca2+ influx is followed by a rapid Ca2+ uptake into the mitochondria via mitochondrial Ca2+ uniporter (MCU) and is driven by a steep mitochondrial membrane potential (ΔΨm) across the inner mitochondrial membrane. Calcium is subsequently released from the mitochondria by a ˜10–100fold slower mitochondrial Na+/Ca2+ exchanger, NCLX. NCLX-mediated mitochondrial Ca2+ removal may be upregulated by (1) an increase in cytosolic Na+, mediated by TRP channels or voltage-sensitive Na+ channels (Nav) at the PM and by (2) PKA-dependent phosphorylation at Ser258 of NCLX, that also prevents ΔΨm-driven allosteric inhibition (3) triggered for example by UCP1-mediated uncoupling. By linking plasmalemmal and mitochondrial Na+ and Ca2+ signals, NCLX is essential in different physiological processes, ranging from insulin secretion to pain sensation. Knockout of NCLX or its impaired activity (for example due to PINK1 deficiency) leads to mitochondrial Ca2+ overload, inhibition of MCU and opening of the mitochondrial permeability transition pore (MPTP), which is a hallmark and preceding event in mitochondrial dysfunction linked to ischemic or neurodegenerative syndromes.
employing the mitochondrial Na+ reporter CoroNa Red showed that modulation of cytosolic Na+ by the astrocytic glutamate transporters is followed by activation of the mitochondrial Na+/Ca2+ exchanger, leading to a robust Na+ influx into the mitochondria [40]. Recent studies have highlighted a major role of NCLX in astrocytes. The knockdown (KD) of NCLX inhibited the SOC pathway [41]. Consistent with this finding Ca2+ fluxes between SOC channel and mitochondria were much stronger than the ER-mitochondrial Ca2+ transfer in astrocytes [41]. The large fluxes of cytosolic and mitochondrial Na+ in astrocytes underscore the major role of Na+ in the communication between cell membrane, SOC and the mitochondria. Analysis of mitochondrial Na+ movements in cardiac cells also indicated that the mitochondrial Na+/Ca2+ exchanger is the major route for mitochondrial Na+ influx [42]. Excessive activation of this exchanger was linked to energy shortage encountered in cardiac ischemia. The rise in cytosolic Na+ was shown to strongly activate the mitochondrial Na+/ Ca2+ exchanger, leading to mitochondrial Ca2+ depletion that inhibits the mitochondrial metabolic rate, thus aggravating ATP shortage encountered during ischemic insults [43]. Molecular studies on the interaction of the exchanger with other cellular Na+ routes identified a surprisingly active interaction. For example, in pancreatic β cells, the sustained depolarization leads to a massive Na+ influx via the voltage-gated Na+ channel. This Na+ wave is propagating to the mitochondria via NCLX and powers the mitochondrial Ca2+ shuttling that is initiated by glucose rise. The enhanced Ca2+ shuttling through the mitochondria can reduce the local Ca2+ concentration near the Ca2+ inhibitory site of the LTCC, thus facilitating a prolonged Ca2+ influx through this pathway and leading to enhanced and prolonged insulin secretion [15,44]. A similar role for mitochondrial Ca2+ shuttling powered by the mitochondrial Na+ uptake via NCLX is found in dorsal root ganglion (DRG) pain sensory neurons [17]. Activation of the nociceptor by capsaicin leads to a rise in cytosolic Na+ that is propagated to the mitochondria and enhances mitochondrial Ca2+ shuttling. The KD of NCLX leads to a profound inhibition of DRG neurons firing triggered by capsaicin. TRVP1 electrical activity however can be fully rescued when cytosolic Ca2+ is
buffered by the application of intracellular BAPTA [17]. Thus, by enhancing mitochondrial Ca2+ shuttling, the mitochondrial exchanger prevents the allosteric inhibition of TRPV1 that can be triggered by a local rise of cytosolic Ca2+. Mitochondrial Na+/Ca2+ exchanger can however crosstalk with cell membrane channels through other distinct pathways (Fig. 1). Most notable is the store operated Ca2+ channel, the major Ca2+ influx route in non-excitable cells. Molecular KD of NCLX leads to a profound inhibition of SOC-mediated Ca2+ influx and current [45]. Remarkably, the omission of extracellular Na+ leads to a similar inhibition of SOC activity. Surprisingly, and in contrast to the LTCC and TRPV1, buffering of cytosolic Ca2+ failed to rescue SOC-dependent Ca2+ influx or current. In addition, the KD of NCLX did not affect the interaction between the major SOC components, the ER Ca2+ sensor-Stim1 and the cell membrane Ca2+ channel-Orai1. Store operated Ca2+ influx induced however a mitochondrial redox transient that was abolished upon the KD of NCLX. Analysis of Orai1 mutated at their redox sensitive Cys195 showed that mitochondrial redox transient induced by NCLX is acting via this Orai1 domain [45]. It should be noted that NCLX activity is saturated at ˜15 mM of cytosolic Na+. Subplasmalemmal Ca2+ sequestration by mitochondria has been thought to prevent Ca2+-deactivation of SOC channels [46]. Subplasmalemmal Na+ accumulation has also been described in cells that host SOCE pathway [47]. By subplasmelemmal Na+ accumulation, local NCLX activity could be accelerated at first, but if it crosses the threshold of ˜ 15 mM for cytosolic Na+, NCLX activity could further remain unchanged or even inhibited. Hence, such ion channeling by local Na+-mediated subplasmalemmal inhibition of NCLX activity has been functionally shown as well [46,48].Thus, the Ca2+ and Na+ signals linked to SOC are converging on the mitochondria, leading to a redox signaling that are further controlling SOC activity [45]. Clearly many more physiological phenomena are linked to Na+ signaling crosstalk with the mitochondria. Our ability to identify these novel pathways are severely limited by lack of high-quality Na+ fluorescent reporters. An additional setback to this field was the discontinuation of the mitochondrial Na+ sensor CoroNa Red. Considering 61
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the importance and potential of Na+ signaling, we expect that this probe will be reestablished and other, more advanced Na+ sensors will be developed.
mutation, the expression of the constitutively active NCLX mutation (S258D) enhanced mitochondrial Ca2+ efflux and rescued neurons from mitochondrial Ca2+ overload [57]. A major unresolved question is why mitochondrial Ca2+ overload leads to the inhibition of NCLX. Remarkably, mitochondrial Ca2+ overload is also associated with mild mitochondrial depolarization [58,59]. Recently we have found that even a mild mitochondrial depolarization specifically and allosterically regulates mitochondrial Ca2+ efflux by NCLX [60]. Thus, mild mitochondrial depolarization induced by chemical or biological (UCP1) uncouplers is sufficient to profoundly inhibit NCLX activity. Analysis of NCLX sequence showed that allosteric regulation of NCLX by ΔΨm can be mediated by two clusters of positively charged residues. Importantly, the allosteric regulation of NCLX by ΔΨm could be switched off by PKA phosphorylation of NCLX at the Ser258 site (Fig. 1). Therefore this site can be regarded as a safety switch to prevent a vicious cycle of mitochondrial Ca2+ overload encountered in ischemic or neurodegenerative syndromes such as the PINK1 mutation [60]. Such dual and allosteric regulation of NCLX by ΔΨm and phosphorylation can link between the metabolic state and mitochondrial Ca2+ signaling. Future studies are required to determine if this mode of regulation can be harnessed as a therapeutic strategy to prevent cardiac or brain damage linked to ischemic or neurodegenerative diseases. Further studies are also required to identify the physiological role and pathophysiological implication of NCLX phosphorylation on its many other sites.
5. Regulation of the mitochondrial Na+/Ca2+ exchanger by ions and proteins Studies carried out in isolated cardiac mitochondria and mitoplasts indicated that the exchanger is strongly regulated by Ca2+ [49]. A rise in Ca2+ concentration was followed by a partial inhibition of the exchange activity. The NCX members of the superfamily are strongly and allosterically regulated by Ca2+ that binds to a Ca2+ binding domains (CBDs) termed CBD1 and CBD 2 [50,51]. NCLX has a much smaller regulatory domain with no apparent CBD domains. In fact, there are no such Ca2+ binding domains in any part of NCLX sequence. Calcium may also act indirectly on the exchanger, by activating for example Calpain that will cleave it, or by affecting the ΔΨm and MCU activity. It is therefore unclear if Ca2+ is allosterically acting directly or indirectly on the exchanger. Other studies suggest that the exchanger is regulated by pH, reaching an optimal activity at cytosolic pH of 7.6 [52]. The exchanger was activated by nigericin at neutral pH range, but application of nigericin at the acidic range inhibited the exchanger. At an acidic pH, nigericin can increase the matrix Na+ concentration, thus diminishing the transmitochondrial Na+ gradient. In addition, Ca2+ buffering in the matrix is strongly pH-dependent and it will be therefore important to exclude that pH is acting by changing the matrix Ca2+ or Na+ concentrations. Several studies indicated that the mitochondrial Na+/Ca2+ exchanger is regulated by proteins and kinases. For example Stomatin-like protein 2 (SLP-2), an inner mitochondrial membrane protein, inhibited mitochondrial Na+- dependent Ca2+ removal, while depletion of the protein accelerated this process [53]. Although the exact function of this protein is unknown, the authors suggested that it may act as chaperonin modulating the mitochondrial distribution or expression of the mitochondrial exchanger. Future studies monitoring the expression and distribution of NCLX as a function of SLP-2 expression are required to address this hypothesis. In another study, tetanic stimulation of Xenophous motor neurons induced a strong potentiation of transmitter release that was induced by Na+-dependent mitochondrial Ca2+ release [54]. This effect was prevented by PKC inhibitor, indicating that PKC may upregulate NCLX activity in inducing this effect. In silico analysis of NCLX sequence failed to identify a clear PKC consensus site. Thus it remained to be determined if PKC effect on NCLX is direct or indirect. Mitochondrial Ca2+ overload, a hallmark of ischemic or neurodegenerative syndromes is manifested by a chronically prolonged rise in mitochondrial free Ca2+. By employing a cellular model of Parkinson’s disease related to mutations in PTEN-induced putative kinase 1 (PINK1), it was shown that the mitochondrial Ca2+ overload in cells deficient of PINK1 is linked to a failure of mitochondrial Ca2+ efflux [55]. Further studies, employing Na+ and Ca2+ imaging suggested that reduced mitochondrial Ca2+ efflux is linked to impaired activity of the mitochondrial Na+/Ca2+ exchanger. Subsequent study indicated that NCLX could be linked to this PINK1 effect [56]. However, PINK1 did not directly interact with NCLX nor it affected its expression. It was however found that NCLX activity could be fully rescued by PKA activation. This rescuing effect was mediated by PKA phosphorylation of NCLX on a serine residue 258 (Ser258) found on NCLX regulatory domain (Fig. 1). Mutation of Ser258 to Asp (S258D) or to Ala (S258A) triggered a PKA-independent activation or inactivation of NCLX, respectively. Mutations in leucine rich repeat kinase 2 (LRRK2) are linked to late onset of familiar Parkinson diseases. Analysis of mitochondrial Ca2+ show that these mutations are linked to rise in the expression of mitochondrial Ca2+ influx components MCU and MICU1, leading to enhanced accumulation of mitochondrial Ca2+ [57]. Although the expression of NCLX was not altered in cell expressing the LRRK2
6. Knockout model of NCLX The use of knockout (KO) models is a powerful strategy to address physiological and pathophysiological role of genes. A major surprise that arose when MCU KO mice were analyzed was their very mild phenotype [61,62]. Despite the lack of mitochondrial Ca2+ influx, mice manifested a mild muscular weakness and were susceptible to ischemiainduced brain damage. A conditional KO showed a somewhat more severe phenotype, but still raised the puzzling question of how impairment of a central physiological process such as mitochondrial Ca2+ influx triggers a surprisingly mild phenotype [63,64]. A strong support for the critical role of mitochondrial Ca2+ was demonstrated by a conditional cardiac KO of NCLX [65]. This study showed that tamoxifen-induced deletion of NCLX in adult mouse hearts causes sudden death. Lethality was linked to severe myocardial tissue damage and dysfunction, leading to heart failure. At a cellular level, cardiac pathology was attributed to mitochondrial Ca2+ overload, leading to oxygen radical production and necrotic cell damage that could be partially rescued by inhibition of the mitochondrial permeability transition pore (Fig. 1). On the other hand, overexpression of NCLX in the mouse heart enhanced clearance of mitochondrial Ca2+, and protected against ischemia-induced heart damage [65]. The cause for the striking differences between the MCU and NCLX KO model is not clear, but may be attributed to several reasons. Calcium influx into the mitochondria is largely powered by the steep ΔΨm. Therefore, every pathway with even a partial Ca2+ permeability may compensate for MCU deficiency. Indeed, several studies indicated that Ca2+ influx persists in MCU-deficient neurons and other cells type although the molecular identity of a channel that can carry this function is unknown [66]. NCLX on the other hand has to carry a more complex Na+-dependent pumping of Ca2+ that is required for mitochondrial Ca2+ removal. NCLX is a distinct member of the phylogenic tree of the Na+/Ca2+ exchanger superfamily and while other NCX members were proposed to act on the outer mitochondrial membrane, there is no molecular indication for another Na+/Ca2+ exchanger in the inner mitochondrial membrane. Activity of H+/Ca2+ exchanger has been documented, but it’s generally thought to be slower and less ubiquitous then NCLX activity. In addition, the activity of NCLX is 10–100 slower than MCU and thus constitutes a rate-limiting step in the mitochondrial Ca2+ transient [67]. All in all, the dramatic phenotype of NCLX KO 62
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LTCC similarly to the mitochondrial Na+/Ca2+ exchanger [75]. Importantly, the neuroprotective effect attributed to CGP37157 block of the exchanger was found to be largely related to the block of the LTCC that prevented cytosolic, followed by mitochondrial Ca2+ overload and neuronal toxicity. However, studies on neurodegenerative Parkinson’s disease cellular model indicated that activation of NCLX is neuroprotective in PINK1-deficient neurons [60] and that expression of a constitutively active NCLX mutated at its regulatory domain confers protection in LRRK2 model [57]. Similarly, frataxin deficiency, a cell model of Friedrich’s ataxia leads to mitochondrial swelling in cardiomyocytes and compromises DRG neurons survival [76]. These processes are linked to reduced expression of NCLX that can be restored by Ca2+ chelating agent. Remarkably, the reduction of NCLX may be linked to enhanced degradation of this exchanger by Calpain [31,76]. Thus the role of NCLX during brain ischemia should be reexamined using a more selective molecular model, for example a conditional KO model such as the one used to interrogate the role of NCLX in the heart. Recent studies showed that C. elegans NCX homologue is also expressed in mitochondria and is important for proper neuronal development [77]. The KO of NCX in this model was followed by impaired axonal guidance. In the mammalian brain, Ca2+ conducting TRPCs are critical for proper axonal pruning and guidance [78]. It will be therefore of interest to determine if mitochondrial Ca2+, through the activity of NCLX is playing a similar role in the mammalian brain.
indicates that this exchanger is a very important therapeutic target in ischemic or neurodegenerative diseases. 7. NCLX and ER Ca2+ signaling Mitochondrial-ER Ca2+ signaling is mediated by many components that control and fine tune Ca2+ shuttling between these two organelles. A well-controlled crosstalk between these domains is crucial not only for Ca2+ shuttling but also for the energetic status of the cell. The mitochondria can be crucial for Ca2+ recycling back into the ER and serve as a relay domain in communicating ER Ca2+ response to the cytosol. Molecular studies focusing on NCLX revealed a complex role of this exchanger in ER Ca2+ signaling. In HEK cells, the KO of NCLX had a small effect on Ca2+ refilling [8]. In contrast, in B lymphocytes the KO of NCLX was followed by a profound depletion of ER Ca2+, indicating that the KO of this exchanger reduced basal content of the ER [68]. Consistent with this effect, antigen receptor Ca2+ responses were impaired. Remarkably, this study showed that ER Ca2+ uptake was severely impaired following NCLX KO underscoring the major role of this exchanger in ER Ca2+ recycling. Similarly, in cardiac cell line the KD of NCLX reduced the sarcoplasmic reticulum (SR) Ca2+ content and as a result changed the length of spontaneous Ca2+ oscillations and action potential generations [69]. The molecular basis for NCLX induced oscillations was recently interrogated in mast cells with depolarized, deenergized mitochondria, a scenario that is found for example during ischemic insults [70]. Under these conditions, NCLX is becoming dominant in generating mitochondrial Ca+ oscillations by intermittently acting in its reverse and forward mode, thus triggering transient cycles of mitochondrial Ca2+ uptake and removal. The oscillatory effect of NCLX is controlled by functional interaction with Mitofusin, found on the outer mitochondrial membrane [70]. The complex role of NCLX in controlling ER-mitochondrial interaction points to a fundamental molecular mechanism that can control the heterogeneous behavior. A recent study on heart tissue interrogated the distribution of NCLX in SR-bound versus SR-unbound mitochondria [71]. By separating these two mitochondrial populations by centrifugation, they then compared their NCLX content. Remarkably, NCLX expression in SR-associated mitochondria was much lower, while MCU was higher. Accordingly the Na+-dependent mitochondrial Ca2+ efflux was reduced in SR-associated versus non SR-associated mitochondria. The elevated Ca2+ of NCLX-deficient SR-tethered mitochondria was associated with an enhanced mitochondrial metabolic activity [71]. Thus this study provided a molecular proof for a polarized mitochondrial distribution of NCLX, explaining the complex and heterogeneous crosstalk of NCLX with ER in various cell types. An intriguing question is what controls and shapes such NCLX distribution. Interestingly, NCLX can be degraded by the Ca2+ activated protease Calpain. It would be interesting to determine if Calpain inhibitor disrupts the polarized NCLX distribution, for example in cardiac tissue.
9. Conclusions and future directions The molecular identification of the mitochondrial Na+/ Ca2+ exchanger NCLX was followed by major progress in our understating of the mode of its regulation and identification of new crosstalk pathways between mitochondria and other cellular domains. It also provided the tools for interrogating physiological role of Ca2+ shuttling players and pathophysiological implication when they are deficient at a cellular level and in vivo. This discovery also opened the door for many fundamental and intriguing challenges that lay ahead: 1) Blockers and agonists of NCLX are required to study its physiological role and in ameliorating the severe mitochondrial Ca2+ overload when it is deficient. Available blockers of this exchanger are nonselective and a screen for more selective one is required. In addition, a new screen will identify NCLX agonists that will reactivate it when its activity is impaired, for example in ischemic or neurodegenerative syndromes. 2) In a search for new NCLX modulating agent, the 3D model could be used in conjunction with molecular dynamics analysis. However, these 3D models are not sufficiently accurate and effort should be made to obtain a high resolution 3D structure of NCLX, based on cryo-EM or high quality crystals. 3) Important progress was made to establish the intracellular distribution of NCLX and its association with other cellular compartments, most notably the ER. Quality of commercially available NCLX antibodies, as well of MCU antibodies, is however not sufficient for a high-resolution immunohistochemical analysis. Generation of antibodies for membrane proteins is indeed often difficult. Therefore, another approach would be to generate a mouse model expressing tagged form of NCLX, using for example CRISPR/ Cas9 gene editing. This model would facilitate the interrogation of NCLX distribution at a tissue and subcellular levels. 4) Conditional cardiac-specific KO mouse model provided important insight on essential role of NCLX in cardiac function. The use of this strategy can identify the role of mitochondrial Ca2+ signaling in function and regulation of other organs and systems too, such as brain, the immune and secretory systems. 5) Identification of human mutation of NCLX can pinpoint its pathophysiological relevance and open the way for a therapeutic
8. Disease model and physiological activity linked to NCLX Early studies employing brain ischemia model and mitochondrial exchanger inhibitor CGP37157 found that blocking of the mitochondrial Na+/Ca2+ exchanger plays a protective effect, both in cultured neurons and brain slices [72,73]. While CGP37157 was considered the most selective and potent inhibitor of the exchanger, many subsequent studies found that, perhaps not very surprisingly, this benzodiazepine compound interacts and modulates the activity of many other Ca2+ channels and transporters. A notable example was observed in the pancreatic β cells. In vivo studies employing CGP37157 claimed that insulin secretion can be augmented by blocking the mitochondrial exchanger. Subsequent studies showed that CGP37157 is as potent in blocking LTCC, which is also critical in controlling insulin secretion [74]. A recent study which examined the selectivity of CGP37157 in neurons confirmed that this compound is indeed effective in blocking 63
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approach. The availability of major genetic banks would enable the screen and identification of such mutations and to understand their medical implication.
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Review
Functional properties and mode of regulation of the mitochondrial Na+/ Ca2+ exchanger, NCLX Marko Kostic, Israel Sekler
T
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Department of Physiology and Cell Biology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel
A R T I C LE I N FO
A B S T R A C T
Keywords: NCLX MCU Mitochondrial Ca2+ signaling Ca2+ and neurodegeneration
Mitochondrial Ca2+ transient is the earliest discovered organellar Ca2+ signaling pathway. It consist of a Ca2+ influx, mediated by mitochondrial Ca2+ uniporter (MCU), and mitochondrial Ca2+ efflux mediated by a Na+/ Ca2+ exchanger (NCLX). Mitochondrial Ca2+ signaling machinery plays a fundamental role in linking metabolic activity to cellular Ca2+ signaling, and in controlling local Ca2+ concertation in distinct cellular compartments. Impaired balance between mitochondrial Ca2+ influx and efflux leads to mitochondrial Ca2+ overload, an early and key event in ischemic or neurodegenerative syndromes. Molecular identification of NCLX and MCU happened only recently. Surprisingly, MCU knockout yielded a relatively mild phenotype while conditional knockout of NCLX led to a rapid fatal heart failure. Here we will focus on recent functional and molecular studies on NCLX structure and its mode of regulation. We will describe the unique crosstalk of this exchanger with Na+ and Ca2+ signaling pathways in the cell membrane and the endoplasmic reticulum, and with protein kinases that posttranslationally modulate NCLX activity. We will critically compare selectivity of pharmacological blockers versus molecular control of NCLX expression and activity. Finally we will discuss why this exchanger is essential for survival and can serve as an attractive therapeutic target.
1. Introduction Cellular Ca2+ signaling is of central importance for numerous cellular functions, echoing the famous saying of the late Nobel laureate Otto Loewi: "Ja, Kalzium, das ist alles" (Yes, calcium is everything). Mitochondria were among the earliest cellular organelles investigated for Ca2+ signaling. Early studies conducted in isolated mitochondria during 1960s showed that these organelles are capable of taking up large amounts of Ca2+ [1,2]. These early studies also indicated that the mitochondrial Ca2+ uptake is powered by, and strongly dependent on, the steep mitochondrial membrane potential (ΔΨm), as mitochondrial depolarization by protonophores or uncouplers abolished mitochondrial Ca2+ uptake. Subsequent studies have found that following the influx of Ca2+ into the mitochondria, Ca2+ could be pumped out of mitochondria primarily by a Na+-dependent process [3]. The focus and interest in mitochondrial Ca2+ signaling however waned with the development of the first fluorescent probes for cytosolic Ca2+. Analysis of Ca2+ changes using these probes suggested that cytosolic Ca2+ spikes in most non-excitatory cell types are reaching concentrations of hundreds of nM at most. These values were much smaller than the apparent affinity for mitochondrial Ca2+ uptake (tens of μM Ca2+ concentration) that was previously determined in isolated ⁎
mitochondria, thus indicating that mitochondrial Ca2+ uptake is of no physiological significance. These sober conclusions, taken together with the emergence of the endoplasmic reticulum (ER) as a major Ca2+storing organelle in the cell, led to a shift of interest from mitochondria to ER. For about 20 years, the mitochondrial Ca2+ signaling was largely abandoned. The change came in the 1990s by key experiments performed by Rizzuto and Pozzan. They were the first to use geneticallyencoded Ca2+ probes-the aequorins that were targeted to the mitochondrial matrix. In contrast to the results obtained in isolated mitochondria, many of the Ca2+ signals triggered in the ER and cytosol in cells were effectively propagated into the mitochondria [4,5]. These landmark experiments revived the discipline of mitochondrial Ca2+ signaling. The discrepancy between the in vitro and cellular results was resolved by subsequent demonstration that Ca2+ rise within the cell is not homogenously distributed, but is order of magnitudes higher in the so-called “hot spots”-cellular domains at mitochondria found in the vicinity of the plasma membrane or at ER-junctions [6]. Subsequent studies confirmed the role of specialized interaction junction between the mitochondria and ER where the flow of Ca2+ is particularly efficient and intense [7]. The second landmark breakthrough that occurred twenty years later were the molecular discoveries of the mitochondrial Na+/ Ca2+ exchanger, NCLX [8], and shortly afterwards of the
Corresponding author. E-mail address: [email protected] (I. Sekler).
https://doi.org/10.1016/j.semcdb.2019.01.009 Received 11 November 2018; Received in revised form 14 January 2019; Accepted 14 January 2019 Available online 30 January 2019 1084-9521/ © 2019 Elsevier Ltd. All rights reserved.
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mitochondrial Ca2+ uniporter-MCU [9,10]. The earlier and recent studies have identified several key roles for mitochondrial Ca2+ signaling: 1) Ca2+ rise is upregulating the activity of at least three enzymes in the Krebs cycle and that of the Fo/F1 ATPase [11–13]. Therefore, mitochondrial Ca2+ transient is an essential link between cytosolic Ca2+ signaling and changes in energy demand required for the activation of numerous downstream kinases and motor proteins; 2) In addition, the mitochondrial network is not static but is moving toward cell regions where Ca2+ signaling is particularly intense, predominantly at the vicinity of the ER and plasma membrane, and regulates the local Ca2+ concentrations at these domains [14]. Because major Ca2+ channels at the cell membrane and ER are allosterically regulated by cytosolic Ca2+, the control of cytosolic Ca2+ by the mitochondria strongly regulates activity of these channels. Most notable examples are the L-type Ca2+ channel (LTCC) [15], the ryanodine receptor (RyR) [16], transient receptor potential cation channel subfamily V member 1 (TRPV1) [17] or the store-operated Ca2+ channel (SOC) [18]; 3) In major pathophysiological syndromes, such as brain and cardiac ischemia or in many neurodegenerative syndromes, mitochondrial Ca2+ transient is impaired, often leading to the toxic accumulation of mitochondrial free Ca2+, termed mitochondrial Ca2+ overload [19–23]. Mitochondrial Ca2+ overload is induced by imbalance between mitochondrial Ca2+ uptake and removal, and often leads to mitochondrial swelling, opening of the mitochondrial permeability transition pore and initiation of apoptotic or necrotic cell death [24,25]. In this review we will focus on the mitochondrial Ca2+ removal, in particular on NCLX.
of the Na+/Ca2+ superfamily that diverged early on from the other two families, the NCX and NCKX. Surprisingly, in contrast to all other known members of the superfamily, it conducted an active Li+/Ca2+ exchange as well. Based on this unique Li+-dependent Ca2+ transport we termed it Na+/Li+/Ca2+ exchanger (NCLX) and reasoned that it may be linked to the long sought mitochondrial Na+/Ca2+ exchanger [32]. Western blot analysis revealed that NCLX was found in the mitochondrial fraction, and immunogold electron microscopy (EM) analysis showed that NCLX is found in the inner mitochondrial membrane. Functional analysis of mitochondrial Ca2+ transport showed that overexpression of NCLX enhanced, while silencing of NCLX impaired mitochondrial Ca2+ efflux [8]. Moreover, Li+ and Na+ fully supported mitochondrial Ca2+ efflux mediated by NCLX. All these results converged upon conclusion that NCLX is the long sought mitochondrial Na+/Ca2+ exchanger. 3. Catalytic site of NCLX The 3D structure of the archaebacterial Na+/Ca2+ exchanger NCX_Mj and the homologous H+/Ca2+ exchanger, CAX_Af provided an important handle to study NCLX transport sites [33–35]. A 3D model of NCLX using ROBETTA server indicated that despite the modest sequence homology of NCLX with its archaebacterial homologues they share a strikingly similar arrangement of the Na+ and Ca2+ binding sites [36]. The unique ability of NCLX to conduct both Na+/Ca2+ and Li+/Ca2+ transport makes it a good system to analyze monovalent cation selectivity. Mutagenesis screening showed that mutations in the α1 and α2 catalytic domains confer Li+ over Na+ selectivity. However, only mutation in a single residue of the α2 catalytic domain (D471) conferred Na+ over Li+ selectivity [36]. Another study that modeled NCLX according to NCX_Mj found that despite the strong 3D similarities of the Na+ and Ca2+ binding sites, nine coordinating residues of NCLX are distinct [37]. Replacing these residues on the backbone of Na+ selective NCX_Mj was followed by NCLX-like Li+-dependent Ca2+ exchange of the NCX Mj, thus indicating that this set of residues is required to confer the unique monovalent cation selectivity of NCLX. Future 3D structures of NCLX based on cryo EM or crystal structure will be required to determine precise NCLX ion transport site. In addition, it is important to note that NCX_Mj and other bacterial homologues lack the regulatory domain that apparently plays a key role in controlling the exchanger activity. The 3D structures of the regulatory domains of both NCX and NCLX are therefore required to understand the structural basis of their mode of regulation of ion transport and how they interact with the transport site.
2. Functional and molecular discovery of the mitochondrial Na+/ Ca2+ exchanger Activity of the mitochondrial Na+/Ca2+ exchanger was first identified in isolated cardiac mitochondria that were loaded with Ca2+ [3]. In the absence of Na+, no Ca2+ efflux was observed. However, when Na+ was added, it triggered a rapid mitochondrial Ca2+ efflux. A remarkable feature of the mitochondrial exchanger was that Li+ could replace Na+ in catalyzing mitochondrial Ca2+ efflux. This unique functional trait was vital for later molecular identification of the mitochondrial Na+/Ca2+ exchanger [26]. Subsequent studies found that mitochondrial Na+/Ca2+ exchange is a ubiquitous pathway found in many cell types and critical for many physiological activities. One notable example was found in brown adipose tissue (BAT) during norepinephrine-triggered uncoupled respiration, where Ca2+ that accumulated in BAT mitochondria could be subsequently removed only in the presence of Na+ [27]. Moreover, inhibition of mitochondrial Ca2+ efflux in the absence of Na+ severely impaired uncoupled respiration, underscoring the vital role of the exchanger in controlling the metabolic activity in BAT. Other studies have emphasized the unique divalent cation selectivity of NCLX for Ca2+ over Mg2+, Mn2+ and Ba2+ [3,28]. The use of matrix targeted Na+ dyes, most notably CoroNa Red, confirmed that the Ca2+ efflux mediated by the mitochondrial Na+/Ca2+ exchanger is coupled to mitochondrial Na+ influx. Effort to identify the mitochondrial Na+/Ca2+ exchanger gene started already in 1990s. Polypeptide candidates of ˜60 kDa and ˜110 kDa molecular weights were purified from beef heart [29,30]. Proteoliposmes reconstituted with these exchanger candidates showed Na+- and Li+-dependent Ca2+ efflux. Furthermore, antibody raised against the polypeptides and blockers of this exchanger inhibited Na+and Li+-dependent Ca2+ efflux. Moreover, the exchanger was highly sensitive to degradation by mitochondrial Ca2+-dependent protease μCalpain [31]. The molecular identification of NCLX as the mitochondrial Na+/ 2+ Ca exchanger started when we cloned and functionally analyzed a new member of the Na+/Ca2+ exchanger superfamily. A phylogenic analysis indicated that this gene is a single member of a distinct family
4. Na+-dependent functions of the mitochondrial Na+/Ca2+ exchanger Most of the interest and focus regarding the mitochondrial Na+/ Ca exchanger is about its Ca2+ signaling role. An often-overlooked feature of this transporter is that it transports 3Na+ per 1Ca2+, and is therefore transporting more Na+ ions in than Ca2+ ions out of the mitochondria. The knowledge and interest on the role of Na+ in cell signaling is much more limited than that of Ca2+. The general notion was that changes in transmembrane Na+ gradient are small, largely insignificant and that the major role of Na+ is in controlling the electrical activity in excitable cells. In addition, the quality of cellular fluorescent Na+ reporters is far lower than that of pH- or Ca2+- fluorescent reporters, thus limiting our ability to monitor changes in the concertation of Na+ [38]. Nevertheless, several key and early studies underscored the role of mitochondrial Na+ signaling by the mitochondrial Na+/Ca2+ exchanger. First, analysis of affinity of the exchanger to Na+ showed that it is ˜7 mM, close to the resting level of cytosolic Na+ [30,39]. Thus, even a small change in cytosolic Na+ will have a very strong impact on the rate of the exchanger activity (Fig. 1). Indeed, studies in astrocytes 2+
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Fig. 1. Mode of regulation and physiological/pathophysiological roles of NCLX. Calcium enters the cell by multiple pathways at the plasma membrane (PM), such as store-operated Ca2+ channel (SOC), L-type voltagegated Ca2+ channel (LTCC) or cationic channel TRPV1. Cytosolic Ca2+ influx is followed by a rapid Ca2+ uptake into the mitochondria via mitochondrial Ca2+ uniporter (MCU) and is driven by a steep mitochondrial membrane potential (ΔΨm) across the inner mitochondrial membrane. Calcium is subsequently released from the mitochondria by a ˜10–100fold slower mitochondrial Na+/Ca2+ exchanger, NCLX. NCLX-mediated mitochondrial Ca2+ removal may be upregulated by (1) an increase in cytosolic Na+, mediated by TRP channels or voltage-sensitive Na+ channels (Nav) at the PM and by (2) PKA-dependent phosphorylation at Ser258 of NCLX, that also prevents ΔΨm-driven allosteric inhibition (3) triggered for example by UCP1-mediated uncoupling. By linking plasmalemmal and mitochondrial Na+ and Ca2+ signals, NCLX is essential in different physiological processes, ranging from insulin secretion to pain sensation. Knockout of NCLX or its impaired activity (for example due to PINK1 deficiency) leads to mitochondrial Ca2+ overload, inhibition of MCU and opening of the mitochondrial permeability transition pore (MPTP), which is a hallmark and preceding event in mitochondrial dysfunction linked to ischemic or neurodegenerative syndromes.
employing the mitochondrial Na+ reporter CoroNa Red showed that modulation of cytosolic Na+ by the astrocytic glutamate transporters is followed by activation of the mitochondrial Na+/Ca2+ exchanger, leading to a robust Na+ influx into the mitochondria [40]. Recent studies have highlighted a major role of NCLX in astrocytes. The knockdown (KD) of NCLX inhibited the SOC pathway [41]. Consistent with this finding Ca2+ fluxes between SOC channel and mitochondria were much stronger than the ER-mitochondrial Ca2+ transfer in astrocytes [41]. The large fluxes of cytosolic and mitochondrial Na+ in astrocytes underscore the major role of Na+ in the communication between cell membrane, SOC and the mitochondria. Analysis of mitochondrial Na+ movements in cardiac cells also indicated that the mitochondrial Na+/Ca2+ exchanger is the major route for mitochondrial Na+ influx [42]. Excessive activation of this exchanger was linked to energy shortage encountered in cardiac ischemia. The rise in cytosolic Na+ was shown to strongly activate the mitochondrial Na+/ Ca2+ exchanger, leading to mitochondrial Ca2+ depletion that inhibits the mitochondrial metabolic rate, thus aggravating ATP shortage encountered during ischemic insults [43]. Molecular studies on the interaction of the exchanger with other cellular Na+ routes identified a surprisingly active interaction. For example, in pancreatic β cells, the sustained depolarization leads to a massive Na+ influx via the voltage-gated Na+ channel. This Na+ wave is propagating to the mitochondria via NCLX and powers the mitochondrial Ca2+ shuttling that is initiated by glucose rise. The enhanced Ca2+ shuttling through the mitochondria can reduce the local Ca2+ concentration near the Ca2+ inhibitory site of the LTCC, thus facilitating a prolonged Ca2+ influx through this pathway and leading to enhanced and prolonged insulin secretion [15,44]. A similar role for mitochondrial Ca2+ shuttling powered by the mitochondrial Na+ uptake via NCLX is found in dorsal root ganglion (DRG) pain sensory neurons [17]. Activation of the nociceptor by capsaicin leads to a rise in cytosolic Na+ that is propagated to the mitochondria and enhances mitochondrial Ca2+ shuttling. The KD of NCLX leads to a profound inhibition of DRG neurons firing triggered by capsaicin. TRVP1 electrical activity however can be fully rescued when cytosolic Ca2+ is
buffered by the application of intracellular BAPTA [17]. Thus, by enhancing mitochondrial Ca2+ shuttling, the mitochondrial exchanger prevents the allosteric inhibition of TRPV1 that can be triggered by a local rise of cytosolic Ca2+. Mitochondrial Na+/Ca2+ exchanger can however crosstalk with cell membrane channels through other distinct pathways (Fig. 1). Most notable is the store operated Ca2+ channel, the major Ca2+ influx route in non-excitable cells. Molecular KD of NCLX leads to a profound inhibition of SOC-mediated Ca2+ influx and current [45]. Remarkably, the omission of extracellular Na+ leads to a similar inhibition of SOC activity. Surprisingly, and in contrast to the LTCC and TRPV1, buffering of cytosolic Ca2+ failed to rescue SOC-dependent Ca2+ influx or current. In addition, the KD of NCLX did not affect the interaction between the major SOC components, the ER Ca2+ sensor-Stim1 and the cell membrane Ca2+ channel-Orai1. Store operated Ca2+ influx induced however a mitochondrial redox transient that was abolished upon the KD of NCLX. Analysis of Orai1 mutated at their redox sensitive Cys195 showed that mitochondrial redox transient induced by NCLX is acting via this Orai1 domain [45]. It should be noted that NCLX activity is saturated at ˜15 mM of cytosolic Na+. Subplasmalemmal Ca2+ sequestration by mitochondria has been thought to prevent Ca2+-deactivation of SOC channels [46]. Subplasmalemmal Na+ accumulation has also been described in cells that host SOCE pathway [47]. By subplasmelemmal Na+ accumulation, local NCLX activity could be accelerated at first, but if it crosses the threshold of ˜ 15 mM for cytosolic Na+, NCLX activity could further remain unchanged or even inhibited. Hence, such ion channeling by local Na+-mediated subplasmalemmal inhibition of NCLX activity has been functionally shown as well [46,48].Thus, the Ca2+ and Na+ signals linked to SOC are converging on the mitochondria, leading to a redox signaling that are further controlling SOC activity [45]. Clearly many more physiological phenomena are linked to Na+ signaling crosstalk with the mitochondria. Our ability to identify these novel pathways are severely limited by lack of high-quality Na+ fluorescent reporters. An additional setback to this field was the discontinuation of the mitochondrial Na+ sensor CoroNa Red. Considering 61
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the importance and potential of Na+ signaling, we expect that this probe will be reestablished and other, more advanced Na+ sensors will be developed.
mutation, the expression of the constitutively active NCLX mutation (S258D) enhanced mitochondrial Ca2+ efflux and rescued neurons from mitochondrial Ca2+ overload [57]. A major unresolved question is why mitochondrial Ca2+ overload leads to the inhibition of NCLX. Remarkably, mitochondrial Ca2+ overload is also associated with mild mitochondrial depolarization [58,59]. Recently we have found that even a mild mitochondrial depolarization specifically and allosterically regulates mitochondrial Ca2+ efflux by NCLX [60]. Thus, mild mitochondrial depolarization induced by chemical or biological (UCP1) uncouplers is sufficient to profoundly inhibit NCLX activity. Analysis of NCLX sequence showed that allosteric regulation of NCLX by ΔΨm can be mediated by two clusters of positively charged residues. Importantly, the allosteric regulation of NCLX by ΔΨm could be switched off by PKA phosphorylation of NCLX at the Ser258 site (Fig. 1). Therefore this site can be regarded as a safety switch to prevent a vicious cycle of mitochondrial Ca2+ overload encountered in ischemic or neurodegenerative syndromes such as the PINK1 mutation [60]. Such dual and allosteric regulation of NCLX by ΔΨm and phosphorylation can link between the metabolic state and mitochondrial Ca2+ signaling. Future studies are required to determine if this mode of regulation can be harnessed as a therapeutic strategy to prevent cardiac or brain damage linked to ischemic or neurodegenerative diseases. Further studies are also required to identify the physiological role and pathophysiological implication of NCLX phosphorylation on its many other sites.
5. Regulation of the mitochondrial Na+/Ca2+ exchanger by ions and proteins Studies carried out in isolated cardiac mitochondria and mitoplasts indicated that the exchanger is strongly regulated by Ca2+ [49]. A rise in Ca2+ concentration was followed by a partial inhibition of the exchange activity. The NCX members of the superfamily are strongly and allosterically regulated by Ca2+ that binds to a Ca2+ binding domains (CBDs) termed CBD1 and CBD 2 [50,51]. NCLX has a much smaller regulatory domain with no apparent CBD domains. In fact, there are no such Ca2+ binding domains in any part of NCLX sequence. Calcium may also act indirectly on the exchanger, by activating for example Calpain that will cleave it, or by affecting the ΔΨm and MCU activity. It is therefore unclear if Ca2+ is allosterically acting directly or indirectly on the exchanger. Other studies suggest that the exchanger is regulated by pH, reaching an optimal activity at cytosolic pH of 7.6 [52]. The exchanger was activated by nigericin at neutral pH range, but application of nigericin at the acidic range inhibited the exchanger. At an acidic pH, nigericin can increase the matrix Na+ concentration, thus diminishing the transmitochondrial Na+ gradient. In addition, Ca2+ buffering in the matrix is strongly pH-dependent and it will be therefore important to exclude that pH is acting by changing the matrix Ca2+ or Na+ concentrations. Several studies indicated that the mitochondrial Na+/Ca2+ exchanger is regulated by proteins and kinases. For example Stomatin-like protein 2 (SLP-2), an inner mitochondrial membrane protein, inhibited mitochondrial Na+- dependent Ca2+ removal, while depletion of the protein accelerated this process [53]. Although the exact function of this protein is unknown, the authors suggested that it may act as chaperonin modulating the mitochondrial distribution or expression of the mitochondrial exchanger. Future studies monitoring the expression and distribution of NCLX as a function of SLP-2 expression are required to address this hypothesis. In another study, tetanic stimulation of Xenophous motor neurons induced a strong potentiation of transmitter release that was induced by Na+-dependent mitochondrial Ca2+ release [54]. This effect was prevented by PKC inhibitor, indicating that PKC may upregulate NCLX activity in inducing this effect. In silico analysis of NCLX sequence failed to identify a clear PKC consensus site. Thus it remained to be determined if PKC effect on NCLX is direct or indirect. Mitochondrial Ca2+ overload, a hallmark of ischemic or neurodegenerative syndromes is manifested by a chronically prolonged rise in mitochondrial free Ca2+. By employing a cellular model of Parkinson’s disease related to mutations in PTEN-induced putative kinase 1 (PINK1), it was shown that the mitochondrial Ca2+ overload in cells deficient of PINK1 is linked to a failure of mitochondrial Ca2+ efflux [55]. Further studies, employing Na+ and Ca2+ imaging suggested that reduced mitochondrial Ca2+ efflux is linked to impaired activity of the mitochondrial Na+/Ca2+ exchanger. Subsequent study indicated that NCLX could be linked to this PINK1 effect [56]. However, PINK1 did not directly interact with NCLX nor it affected its expression. It was however found that NCLX activity could be fully rescued by PKA activation. This rescuing effect was mediated by PKA phosphorylation of NCLX on a serine residue 258 (Ser258) found on NCLX regulatory domain (Fig. 1). Mutation of Ser258 to Asp (S258D) or to Ala (S258A) triggered a PKA-independent activation or inactivation of NCLX, respectively. Mutations in leucine rich repeat kinase 2 (LRRK2) are linked to late onset of familiar Parkinson diseases. Analysis of mitochondrial Ca2+ show that these mutations are linked to rise in the expression of mitochondrial Ca2+ influx components MCU and MICU1, leading to enhanced accumulation of mitochondrial Ca2+ [57]. Although the expression of NCLX was not altered in cell expressing the LRRK2
6. Knockout model of NCLX The use of knockout (KO) models is a powerful strategy to address physiological and pathophysiological role of genes. A major surprise that arose when MCU KO mice were analyzed was their very mild phenotype [61,62]. Despite the lack of mitochondrial Ca2+ influx, mice manifested a mild muscular weakness and were susceptible to ischemiainduced brain damage. A conditional KO showed a somewhat more severe phenotype, but still raised the puzzling question of how impairment of a central physiological process such as mitochondrial Ca2+ influx triggers a surprisingly mild phenotype [63,64]. A strong support for the critical role of mitochondrial Ca2+ was demonstrated by a conditional cardiac KO of NCLX [65]. This study showed that tamoxifen-induced deletion of NCLX in adult mouse hearts causes sudden death. Lethality was linked to severe myocardial tissue damage and dysfunction, leading to heart failure. At a cellular level, cardiac pathology was attributed to mitochondrial Ca2+ overload, leading to oxygen radical production and necrotic cell damage that could be partially rescued by inhibition of the mitochondrial permeability transition pore (Fig. 1). On the other hand, overexpression of NCLX in the mouse heart enhanced clearance of mitochondrial Ca2+, and protected against ischemia-induced heart damage [65]. The cause for the striking differences between the MCU and NCLX KO model is not clear, but may be attributed to several reasons. Calcium influx into the mitochondria is largely powered by the steep ΔΨm. Therefore, every pathway with even a partial Ca2+ permeability may compensate for MCU deficiency. Indeed, several studies indicated that Ca2+ influx persists in MCU-deficient neurons and other cells type although the molecular identity of a channel that can carry this function is unknown [66]. NCLX on the other hand has to carry a more complex Na+-dependent pumping of Ca2+ that is required for mitochondrial Ca2+ removal. NCLX is a distinct member of the phylogenic tree of the Na+/Ca2+ exchanger superfamily and while other NCX members were proposed to act on the outer mitochondrial membrane, there is no molecular indication for another Na+/Ca2+ exchanger in the inner mitochondrial membrane. Activity of H+/Ca2+ exchanger has been documented, but it’s generally thought to be slower and less ubiquitous then NCLX activity. In addition, the activity of NCLX is 10–100 slower than MCU and thus constitutes a rate-limiting step in the mitochondrial Ca2+ transient [67]. All in all, the dramatic phenotype of NCLX KO 62
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LTCC similarly to the mitochondrial Na+/Ca2+ exchanger [75]. Importantly, the neuroprotective effect attributed to CGP37157 block of the exchanger was found to be largely related to the block of the LTCC that prevented cytosolic, followed by mitochondrial Ca2+ overload and neuronal toxicity. However, studies on neurodegenerative Parkinson’s disease cellular model indicated that activation of NCLX is neuroprotective in PINK1-deficient neurons [60] and that expression of a constitutively active NCLX mutated at its regulatory domain confers protection in LRRK2 model [57]. Similarly, frataxin deficiency, a cell model of Friedrich’s ataxia leads to mitochondrial swelling in cardiomyocytes and compromises DRG neurons survival [76]. These processes are linked to reduced expression of NCLX that can be restored by Ca2+ chelating agent. Remarkably, the reduction of NCLX may be linked to enhanced degradation of this exchanger by Calpain [31,76]. Thus the role of NCLX during brain ischemia should be reexamined using a more selective molecular model, for example a conditional KO model such as the one used to interrogate the role of NCLX in the heart. Recent studies showed that C. elegans NCX homologue is also expressed in mitochondria and is important for proper neuronal development [77]. The KO of NCX in this model was followed by impaired axonal guidance. In the mammalian brain, Ca2+ conducting TRPCs are critical for proper axonal pruning and guidance [78]. It will be therefore of interest to determine if mitochondrial Ca2+, through the activity of NCLX is playing a similar role in the mammalian brain.
indicates that this exchanger is a very important therapeutic target in ischemic or neurodegenerative diseases. 7. NCLX and ER Ca2+ signaling Mitochondrial-ER Ca2+ signaling is mediated by many components that control and fine tune Ca2+ shuttling between these two organelles. A well-controlled crosstalk between these domains is crucial not only for Ca2+ shuttling but also for the energetic status of the cell. The mitochondria can be crucial for Ca2+ recycling back into the ER and serve as a relay domain in communicating ER Ca2+ response to the cytosol. Molecular studies focusing on NCLX revealed a complex role of this exchanger in ER Ca2+ signaling. In HEK cells, the KO of NCLX had a small effect on Ca2+ refilling [8]. In contrast, in B lymphocytes the KO of NCLX was followed by a profound depletion of ER Ca2+, indicating that the KO of this exchanger reduced basal content of the ER [68]. Consistent with this effect, antigen receptor Ca2+ responses were impaired. Remarkably, this study showed that ER Ca2+ uptake was severely impaired following NCLX KO underscoring the major role of this exchanger in ER Ca2+ recycling. Similarly, in cardiac cell line the KD of NCLX reduced the sarcoplasmic reticulum (SR) Ca2+ content and as a result changed the length of spontaneous Ca2+ oscillations and action potential generations [69]. The molecular basis for NCLX induced oscillations was recently interrogated in mast cells with depolarized, deenergized mitochondria, a scenario that is found for example during ischemic insults [70]. Under these conditions, NCLX is becoming dominant in generating mitochondrial Ca+ oscillations by intermittently acting in its reverse and forward mode, thus triggering transient cycles of mitochondrial Ca2+ uptake and removal. The oscillatory effect of NCLX is controlled by functional interaction with Mitofusin, found on the outer mitochondrial membrane [70]. The complex role of NCLX in controlling ER-mitochondrial interaction points to a fundamental molecular mechanism that can control the heterogeneous behavior. A recent study on heart tissue interrogated the distribution of NCLX in SR-bound versus SR-unbound mitochondria [71]. By separating these two mitochondrial populations by centrifugation, they then compared their NCLX content. Remarkably, NCLX expression in SR-associated mitochondria was much lower, while MCU was higher. Accordingly the Na+-dependent mitochondrial Ca2+ efflux was reduced in SR-associated versus non SR-associated mitochondria. The elevated Ca2+ of NCLX-deficient SR-tethered mitochondria was associated with an enhanced mitochondrial metabolic activity [71]. Thus this study provided a molecular proof for a polarized mitochondrial distribution of NCLX, explaining the complex and heterogeneous crosstalk of NCLX with ER in various cell types. An intriguing question is what controls and shapes such NCLX distribution. Interestingly, NCLX can be degraded by the Ca2+ activated protease Calpain. It would be interesting to determine if Calpain inhibitor disrupts the polarized NCLX distribution, for example in cardiac tissue.
9. Conclusions and future directions The molecular identification of the mitochondrial Na+/ Ca2+ exchanger NCLX was followed by major progress in our understating of the mode of its regulation and identification of new crosstalk pathways between mitochondria and other cellular domains. It also provided the tools for interrogating physiological role of Ca2+ shuttling players and pathophysiological implication when they are deficient at a cellular level and in vivo. This discovery also opened the door for many fundamental and intriguing challenges that lay ahead: 1) Blockers and agonists of NCLX are required to study its physiological role and in ameliorating the severe mitochondrial Ca2+ overload when it is deficient. Available blockers of this exchanger are nonselective and a screen for more selective one is required. In addition, a new screen will identify NCLX agonists that will reactivate it when its activity is impaired, for example in ischemic or neurodegenerative syndromes. 2) In a search for new NCLX modulating agent, the 3D model could be used in conjunction with molecular dynamics analysis. However, these 3D models are not sufficiently accurate and effort should be made to obtain a high resolution 3D structure of NCLX, based on cryo-EM or high quality crystals. 3) Important progress was made to establish the intracellular distribution of NCLX and its association with other cellular compartments, most notably the ER. Quality of commercially available NCLX antibodies, as well of MCU antibodies, is however not sufficient for a high-resolution immunohistochemical analysis. Generation of antibodies for membrane proteins is indeed often difficult. Therefore, another approach would be to generate a mouse model expressing tagged form of NCLX, using for example CRISPR/ Cas9 gene editing. This model would facilitate the interrogation of NCLX distribution at a tissue and subcellular levels. 4) Conditional cardiac-specific KO mouse model provided important insight on essential role of NCLX in cardiac function. The use of this strategy can identify the role of mitochondrial Ca2+ signaling in function and regulation of other organs and systems too, such as brain, the immune and secretory systems. 5) Identification of human mutation of NCLX can pinpoint its pathophysiological relevance and open the way for a therapeutic
8. Disease model and physiological activity linked to NCLX Early studies employing brain ischemia model and mitochondrial exchanger inhibitor CGP37157 found that blocking of the mitochondrial Na+/Ca2+ exchanger plays a protective effect, both in cultured neurons and brain slices [72,73]. While CGP37157 was considered the most selective and potent inhibitor of the exchanger, many subsequent studies found that, perhaps not very surprisingly, this benzodiazepine compound interacts and modulates the activity of many other Ca2+ channels and transporters. A notable example was observed in the pancreatic β cells. In vivo studies employing CGP37157 claimed that insulin secretion can be augmented by blocking the mitochondrial exchanger. Subsequent studies showed that CGP37157 is as potent in blocking LTCC, which is also critical in controlling insulin secretion [74]. A recent study which examined the selectivity of CGP37157 in neurons confirmed that this compound is indeed effective in blocking 63
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approach. The availability of major genetic banks would enable the screen and identification of such mutations and to understand their medical implication.
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