Characterization of Mg Inhibition of Mitochondrial Ca Uptake by a Mechanistic Model of Mitochondrial Ca Uniporter

Ca²⁺ is an important regulatory ion and alteration of mitochondrial Ca²⁺ homeostasis can lead to cellular dysfunction and apoptosis. Ca²⁺ is transported into respiring mitochondria via the Ca²⁺ uniporter, which is known to be inhibited by Mg²⁺. This uniporter-mediated mitochondrial Ca²⁺ transport is also shown to be influenced by inorganic phosphate (Pi). Despite a large number of experimental studies, the kinetic mechanisms associated with the Mg²⁺ inhibition and Pi regulation of the uniporter function are not well established. To gain a quantitative understanding of the effects of Mg²⁺ and Pi on the uniporter function, we developed here a mathematical model based on known kinetic properties of the uniporter and presumed Mg²⁺ inhibition and Pi regulation mechanisms. The model is extended from our previous model of the uniporter that is based on a multistate catalytic binding and interconversion mechanism and Eyring's free energy barrier theory for interconversion. The model satisfactorily describes a wide variety of experimental data sets on the kinetics of mitochondrial Ca²⁺ uptake. The model also appropriately depicts the inhibitory effect of Mg²⁺ on the uniporter function, in which Ca²⁺ uptake is hyperbolic in the absence of Mg²⁺ and sigmoid in the presence of Mg²⁺. The model suggests a mixed-type inhibition mechanism for Mg²⁺ inhibition of the uniporter function. This model is critical for building mechanistic models of mitochondrial bioenergetics and Ca²⁺ handling to understand the mechanisms by which Ca²⁺ mediates signaling pathways and modulates energy metabolism.     

Biphasic regulation of mitochondrial Ca2+ uptake by cytosolic Ca2+ concentration

A rise in cytosolic Ca(2+) concentration is used as a key activation signal in virtually all animal cells, where it triggers a range of responses including neurotransmitter release, muscle contraction, and cell growth and proliferation [1]. During intracellular Ca(2+) signaling, mitochondria rapidly take up significant amounts of Ca(2+) from the cytosol, and this stimulates energy production, alters the spatial and temporal profile of the intracellular Ca(2+) signal, and triggers cell death [2-10]. Mitochondrial Ca(2+) uptake occurs via a ruthenium-red-sensitive uniporter channel found in the inner membrane [11]. In spite of its critical importance, little is known about how the uniporter is regulated. Here, we report that the mitochondrial Ca(2+) uniporter is gated by cytosolic Ca(2+). Ca(2+) uptake into mitochondria is a Ca(2+)-activated process with a requirement for functional calmodulin. However, cytosolic Ca(2+) subsequently inactivates the uniporter, preventing further Ca(2+) uptake. The uptake pathway and the inactivation process have relatively low Ca(2+) affinities of approximately 10-20 microM. However, numerous mitochondria are within 20-100 nm of the endoplasmic reticulum, thereby enabling rapid and efficient transmission of Ca(2+) release into adjacent mitochondria by InsP(3) receptors on the endoplasmic reticulum. Hence, biphasic control of mitochondrial Ca(2+) uptake by Ca(2+) provides a novel basis for complex physiological patterns of intracellular Ca(2+) signaling.     

Kinetic model for Ca2+-induced permeability transition in energized liver mitochondria discriminates between inhibitor mechanisms

Cytotoxicity associated with pathophysiological Ca(2+) overload (e.g. in stroke) appears mediated by an event termed the mitochondrial permeability transition (mPT). We built and solved a kinetic model of the mPT in populations of isolated rat liver mitochondria that quantitatively describes Ca(2+)-induced mPT as a two-step sequence of pre-swelling induction followed by Ca(2+)-driven, positive feedback, autocatalytic propagation. The model was formulated as two differential equations, each directly related to experimental parameters (Ca(2+) flux/mitochondrial swelling). These parameters were simultaneously assessed using a spectroscopic approach to monitor multiple mitochondrial properties. The derived kinetic model correctly identifies a correlation between initial Ca(2+) concentration and delay interval prior to mPT induction. Within the model's framework, Ru-360 (a ruthenium complex) and Mg(2+) were shown to compete with the Ca(2+)-stimulated initiation phase of mPT induction, consistent with known inhibition at the phenomenological level of the Ca(2+) uniporter. The model further reveals that Mg(2+), but not Ru-360, inhibits Ca(2+)-induced effects on a downstream stage of mPT induction at a site distinct from the uniporter. The analytical approach was then applied to promethazine, an FDA-approved drug previously shown to inhibit both mPT and ischemia-reperfusion injury. Kinetic analysis revealed that promethazine delayed mPT induction in a manner qualitatively distinct from that of lower concentrations of Mg(2+). In summary, we have developed a kinetic model to aid in the quantitative characterization of mPT induction. This model is consistent with/informative about the biochemistry of several mPT inhibitors, and its success suggests that this kinetic approach can aid in the classification of agents or targets that modulate mPT induction.     

The mitochondrial Ca(2+) uniporter

Mitochondrial Ca(2+) uptake plays a fundamental role in the regulation of energy production and cell survival. Under physiological conditions, mitochondrial Ca(2+) uptake occurs by a uniport mechanism driven electrophoretically by the membrane potential created by the respiratory chain. The activity and the biochemical properties of the mitochondrial calcium uniporter (MCU) were extensively characterized for decades but the molecular identity of the channel has remained elusive. Here, we review the recent discovery of the mitochondria Ca(2+) uniporter that represents a groundbreaking result for the molecular understanding of mitochondrial Ca(2+) homeostasis and will provide insight into the role of mitochondrial Ca(2+) deregulation in the pathogenesis of human disorders.     

Molecular identity and functional properties of the mitochondrial na+/ca2+ exchanger

The mitochondrial membrane potential that powers the generation of ATP also facilitates mitochondrial Ca(2+) shuttling. This process is fundamental to a wide range of cellular activities, as it regulates ATP production, shapes cytosolic and endoplasmic recticulum Ca(2+) signaling, and determines cell fate. Mitochondrial Ca(2+) transport is mediated primarily by two major transporters: a Ca(2+) uniporter that mediates Ca(2+) uptake and a Na(+)/Ca(2+) exchanger that subsequently extrudes mitochondrial Ca(2+). In this minireview, we focus on the specific role of the mitochondrial Na(+)/Ca(2+) exchanger and describe its ion exchange mechanism, regulation by ions, and putative partner proteins. We discuss the recent molecular identification of the mitochondrial exchanger and how its activity is linked to physiological and pathophysiological processes.     

Ca2+ -dependent inactivation of the mitochondrial Ca2+ uniporter involves proton flux through the ATP synthase

Stimulation of receptors on the surface of animal cells often evokes cellular responses by raising intracellular Ca(2+) concentration. The rise in cytoplasmic Ca(2+) drives a plethora of processes, including neurotransmitter release, muscle contraction, and cell growth and proliferation. Mitochondria help shape intracellular Ca(2+) signals through their ability to rapidly take up significant amounts of Ca(2+) from the cytosol via the uniporter, a Ca(2+)-selective ion channel in the inner mitochondrial membrane. The uniporter is subject to inactivation, whereby a sustained cytoplasmic Ca(2+) rise prevents further Ca(2+) uptake. In spite of its importance in intracellular Ca(2+) signaling, little is known about the mechanism underlying uniporter inactivation. Here, we report that maneuvers that promote matrix alkalinisation significantly reduce inactivation whereas acidification exacerbates it. We further show that the F(1)F(0)-ATP synthase complex is an important source of protons for inactivation of the uniporter. These findings identify a novel molecular mechanism that regulates the activity of this ubiquitous intracellular Ca(2+) channel, with implications for intracellular Ca(2+) signaling and aerobic ATP production.     

Interactions between spermine and Mg2+ on mitochondrial Ca2+ transport

The effects of the polyamine spermine on the regulation of Ca2+ transport by subcellular organelles from rat liver, heart, and brain were investigated using ion-sensitive minielectrodes and a 45Ca2+ tracer method. Spermine stimulated Ca2+ uptake by mitochondria but not by microsomes. In the presence of spermine, isolated mitochondria could maintain a free extramitochondrial Ca2+ concentration of 0.3-0.2 microM. Stimulation of the initial rates of Ca2+ uptake and 45Ca2+ cycling of mitochondria by spermine shows that this was accomplished through a decrease of the apparent Km for Ca2+ uptake by the Ca2+ uniporter. The half maximally effective concentration of spermine (50 microM) was in the range of physiological concentrations of this polyamine in the cell. Spermidine was five times less effective. Putrescine was ineffective. The stimulation of mitochondrial Ca2+ uptake by spermine was inhibited by Mg2+ in a concentration-dependent manner. However, the diminished contribution of the mitochondria to the regulation of the free extraorganellar Ca2+ concentration could mostly be compensated for by microsomal Ca2+ uptake. Spermine also reversed ruthenium red-induced Ca2+ efflux from mitochondria. It is concluded that spermine is an activator of the mitochondrial Ca2+ uniporter and Mg2+ an antagonist. By this mechanism, the polyamines can confer to the mitochondria an important role in the regulation of the free cytoplasmic Ca2+ concentration in the cell and of the free Ca2+ concentration in the mitochondrial matrix.     

Impaired regulation of brain mitochondria by extramitochondrial Ca2+ in transgenic Huntington disease rats

Huntington disease (HD) is characterized by polyglutamine expansions of huntingtin (htt), but the underlying pathomechanisms have remained unclear. We studied brain mitochondria of transgenic HD rats with 51 glutamine repeats (htt(51Q)), modeling the adult form of HD. Ca(free)(2+) up to 2 mum activated state 3 respiration of wild type mitochondria with glutamate/malate or pyruvate/malate as substrates. Ca(free)(2+) above 2 mum inhibited respiration via cyclosporin A-dependent permeability transition (PT). Ruthenium red, an inhibitor of the mitochondrial Ca(2+) uniporter, did not affect the Ca(2+)-dependent activation of respiration but reduced Ca(2+)-induced inhibition. Thus, Ca(2+) activation was mediated exclusively by extramitochondrial Ca(2+), whereas inhibition was promoted also by intramitochondrial Ca(2+). In contrast, htt(51Q) mitochondria showed a deficient state 3 respiration, a lower sensitivity to Ca(2+) activation, and a higher susceptibility to Ca(2+)-dependent inhibition. Furthermore htt(51Q) mitochondria exhibited a diminished membrane potential stability in response to Ca(2+), lower capacities and rates of Ca(2+) accumulation, and a decreased Ca(2+) threshold for PT in a substrate-independent but cyclosporin A-sensitive manner. Compared with wild type, Ca(2+)-induced inhibition of respiration of htt(51Q) mitochondria was less sensitive to ruthenium red, indicating the involvement of extramitochondrial Ca(2+). In conclusion, we demonstrate a novel mechanism of mitochondrial regulation by extramitochondrial Ca(2+). We suggest that specific regulatory Ca(2+) binding sites on the mitochondrial surface, e.g. the glutamate/aspartate carrier (aralar), mediate this regulation. Interactions between htt(51Q) and distinct targets such as aralar and/or the PT pore may underlie mitochondrial dysregulation leading to energetic depression, cell death, and tissue atrophy in HD.     

Novel role of phospholipase C-delta1: regulation of liver mitochondrial Ca2+ uptake

Mitochondrial Ca2+ (mCa2+) handling is an important regulator of liver cell function that controls events ranging from cellular respiration and signal transduction to apoptosis. Cytosolic Ca2+ enters mitochondria through the ruthenium red-sensitive mCa2+ uniporter, but the mechanisms governing uniporter activity are unknown. Activation of many Ca2+ channels in the cell membrane requires PLC. This activation commonly occurs through phosphitidylinositol-4,5-biphosphate (PIP2) hydrolysis and the production of the second messengers inositol 1,4,5-trisphosphate [I(1,4,5)P3] and 1,2-diacylglycerol (DAG). PIP2 was recently identified in mitochondria. We hypothesized that PLC exists in liver mitochondria and regulates mCa2+ uptake through the uniporter. Western blot analysis with anti-PLC antibodies demonstrated the presence of PLC-delta1 in pure preparations of mitochondrial membranes isolated from rat liver. In addition, the selective PLC inhibitor U-73122 dose-dependently blocked mCa2+ uptake when whole mitochondria were incubated at 37 degrees C with 45Ca2+. Increasing extra mCa2+ concentration significantly stimulated mCa2+ uptake, and U-73122 inhibited this effect. Spermine, a uniporter agonist, significantly increased mCa2+ uptake, whereas U-73122 dose-dependently blocked this effect. The inactive analog of U-73122, U-73343, did not affect mCa2+ uptake in any experimental condition. Membrane-permeable I(1,4,5)P3 receptor antagonists 2-aminoethoxydiphenylborate and xestospongin C also inhibited mCa2+ uptake. Although extra mitochondrial I(1,4,5)P3 had no effect on mCa2+ uptake, membrane-permeable DAG analogs 1-oleoyl-2-acetyl-sn-glycerol and DAG-lactone, which inhibit PLC activity, dose-dependently inhibited mCa2+ uptake. These data indicate that PLC-delta1 exists in liver mitochondria and is involved in regulating mCa2+ uptake through the uniporter.     

Ca2+ homeostasis and exocytosis in carotid glomus cells: role of mitochondria

In oxygen sensing carotid glomus (type 1) cells, the hypoxia-triggered depolarization can be mimicked by mitochondrial inhibitors. We examined the possibility that, other than causing glomus cell depolarization, mitochondrial inhibition can regulate transmitter release via changes in Ca(2+) dynamics. Under whole-cell voltage clamp conditions, application of the mitochondrial inhibitors, carbonyl cyanide m-chlorophenylhydrazone (CCCP) or cyanide caused a dramatic slowing in the decay of the depolarization-triggered Ca(2+) signal in glomus cells. In contrast, inhibition of the Na(+)/Ca(2+) exchanger (NCX), plasma membrane Ca(2+)-ATPase (PMCA) pump or sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) pump had much smaller effects. Consistent with the notion that mitochondrial Ca(2+) uptake is the dominant mechanism in cytosolic Ca(2+) removal, inhibition of the mitochondrial uniporter with ruthenium red slowed the decay of the depolarization-triggered Ca(2+) signal. Hypoxia also slowed cytosolic Ca(2+) removal, suggesting a partial impairment of mitochondrial Ca(2+) uptake. Using membrane capacitance measurement, we found that the increase in the duration of the depolarization-triggered Ca(2+) signal after mitochondrial inhibition was associated with an enhancement of the exocytotic response. The role of mitochondria in the regulation of Ca(2+) signal and transmitter release from glomus cells highlights the importance of mitochondria in hypoxic chemotransduction in the carotid bodies.     

Bax-mediated Ca2+ mobilization promotes cytochrome c release during apoptosis

Previous studies have demonstrated that Ca(2+) is released from the endoplasmic reticulum (ER) in some models of apoptosis, but the mechanisms involved and the functional significance remain obscure. We confirmed that apoptosis induced by some (but not all) proapoptotic stimuli was associated with caspase-independent, BCL-2-sensitive emptying of the ER Ca(2+) pool in human PC-3 prostate cancer cells. This mobilization of ER Ca(2+) was associated with a concomitant increase in mitochondrial Ca(2+) levels, and neither ER Ca(2+) mobilization nor mitochondrial Ca(2+) uptake occurred in Bax-null DU-145 cells. Importantly, restoration of DU-145 Bax expression via adenoviral gene transfer restored ER Ca(2+) release and mitochondrial Ca(2+) uptake and dramatically accelerated the kinetics of staurosporine-induced cytochrome c release, demonstrating a requirement for Bax expression in this model system. In addition, an inhibitor of the mitochondrial Ca(2+) uniporter (RU-360) attenuated mitochondrial Ca(2+) uptake, cytochrome c release, and DNA fragmentation, directly implicating the mitochondrial Ca(2+) changes in cell death. Together, our data demonstrate that Bax-mediated alterations in ER and mitochondrial Ca(2+) levels serve as important upstream signals for cytochrome c release in some examples of apoptosis.     

The alpha-adrenergic-mediated activation of the cardiac mitochondrial Ca2+ uniporter and its role in the control of intramitochondrial Ca2+ in vivo.

Administration of methoxamine (10 microM, 2 min) to perfused rat hearts increased the rate at which subsequently isolated mitochondria accumulated Ca2+. Methoxamine did not change significantly the development of delta phi with time or the basal rates of Ca2+ flux on inhibition of the uniporter with Ruthenium Red. With 200 microM-Pi, the rates of Ca2+ uptake at constant delta phi were unaffected by the small variations in endogenous [Pi] between mitochondrial preparations, and were also unaffected by changes in internal Ca2+ over the approximate range 8-43 nmol of Ca2+/mg. At low internal Ca2+ (about 8 nmol/mg of protein) the rates of Ca2+ uptake at constant delta phi were unaffected by addition of 200 microM-Pi. Under these conditions, the uniporter activity and the uniporter conductance were increased by 38-40% by methoxamine pretreatment. The endogenous Ca2+ content of mitochondria from control heart was about 1.8 nmol of Ca2+/mg of protein. Perfusion with agonist increased the Ca2+ content as follows: 10 microM-methoxamine (2 min), 48%; 1 microM-isoprenaline (2 min), 100%; 1 microM-adrenaline (2 min), 140%. The implications of the data for the adrenergic control of oxidative metabolism by intramitochondrial Ca2+ is discussed.     

Uncoupling protein 3 (UCP3) modulates the activity of Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) by decreasing mitochondrial ATP production

The uncoupling proteins UCP2 and UCP3 have been postulated to catalyze Ca(2+) entry across the inner membrane of mitochondria, but this proposal is disputed, and other, unrelated proteins have since been identified as the mitochondrial Ca(2+) uniporter. To clarify the role of UCPs in mitochondrial Ca(2+) handling, we down-regulated the expression of the only uncoupling protein of HeLa cells, UCP3, and measured Ca(2+) and ATP levels in the cytosol and in organelles with genetically encoded probes. UCP3 silencing did not alter mitochondrial Ca(2+) uptake in permeabilized cells. In intact cells, however, UCP3 depletion increased mitochondrial ATP production and strongly reduced the cytosolic and mitochondrial Ca(2+) elevations evoked by histamine. The reduced Ca(2+) elevations were due to inhibition of store-operated Ca(2+) entry and reduced depletion of endoplasmic reticulum (ER) Ca(2+) stores. UCP3 depletion accelerated the ER Ca(2+) refilling kinetics, indicating that the activity of sarco/endoplasmic reticulum Ca(2+) (SERCA) pumps was increased. Accordingly, SERCA inhibitors reversed the effects of UCP3 depletion on cytosolic, ER, and mitochondrial Ca(2+) responses. Our results indicate that UCP3 is not a mitochondrial Ca(2+) uniporter and that it instead negatively modulates the activity of SERCA by limiting mitochondrial ATP production. The effects of UCP3 on mitochondrial Ca(2+) thus reflect metabolic alterations that impact on cellular Ca(2+) homeostasis. The sensitivity of SERCA to mitochondrial ATP production suggests that mitochondria control the local ATP availability at ER Ca(2+) uptake and release sites.     

Pilocarpine-induced status epilepticus causes N-methyl-D-aspartate receptor-dependent inhibition of microsomal Mg(2+)/Ca(2+) ATPase-mediated Ca(2+) uptake

Status epilepticus is associated with sustained and elevated levels of cytosolic Ca(2+). To elucidate the mechanisms associated with changes of cytosolic Ca(2+) after status epilepticus, this study was initiated to evaluate the effect of pilocarpine-induced status epilepticus on Mg(2+)/Ca(2+) ATPase-mediated Ca(2+) uptake in microsomes isolated from rat cortex, because the Ca(2+) uptake mechanism plays a major role in regulating intracellular Ca(2+) levels. The data demonstrated that the initial rate and overall Ca(2+) uptake in microsomes from pilocarpine treated animals were significantly inhibited compared with those in microsomes from saline-treated control animals. It was also shown that the inhibition of Ca(2+) uptake caused by status epilepticus was not an artifact of increased Ca(2+) release from microsomes, selective isolation of damaged microsomes from the homogenate, or decreased Mg(2+)/Ca(2+) ATPase protein in the microsomes. Pretreatment with the NMDA antagonist dizocilpine maleate blocked status epilepticus-induced inhibition of the initial rate and overall Ca(2+) uptake. The data suggest that inhibition of microsomal Mg(2+)/Ca(2+) ATPase Ca(2+) uptake is involved in NMDA-dependent deregulation of cytosolic Ca(2+) homeostasis associated with status epilepticus.     

Uncoupling proteins 2 and 3 are fundamental for mitochondrial Ca2+ uniport

Mitochondrial Ca(2+) uptake is crucial for the regulation of the rate of oxidative phosphorylation, the modulation of spatio-temporal cytosolic Ca(2+) signals and apoptosis. Although the phenomenon of mitochondrial Ca(2+) sequestration, its characteristics and physiological consequences have been convincingly reported, the actual protein(s) involved in this process are unknown. Here, we show that the uncoupling proteins 2 and 3 (UCP2 and UCP3) are essential for mitochondrial Ca(2+) uptake. Using overexpression, knockdown (small interfering RNA) and mutagenesis experiments, we demonstrate that UCP2 and UCP3 are elementary for mitochondrial Ca(2+) sequestration in response to cell stimulation under physiological conditions - observations supported by isolated liver mitochondria of Ucp2(-/-) mice lacking ruthenium red-sensitive Ca(2+) uptake. Our results reveal a novel molecular function for UCP2 and UCP3, and may provide the molecular mechanism for their reported effects. Moreover, the identification of proteins fundemental for mitochondrial Ca(2+) uptake expands our knowledge of the physiological role for mitochondrial Ca(2+) sequestration.     

Fe(2+) induces a transient Ca(2+) release from rat liver mitochondria

Isolated mitochondria loaded with Ca(2+) and then exposed to Fe(2+) show a transient release of Ca(2+). The magnitude of this response depends on the Ca(2+) loading and the kinetics of the response depends on the concentration of added Fe(2+). We investigated the Fe(2+)-induced Ca(2+) release mechanism by measuring mitochondrial Ca(2+) uptake in the presence of Fe(2+). The presence of Fe(2+) inhibits Ca(2+) uptake two times. Since mitochondria can cycle Ca(2+) across their inner membrane, the suppression of Ca(2+) uptake, but not release, results in an elevation of the extramitochondrial Ca(2+), thereby varying the steady state. The transient release of Ca(2+) initially observed from mitochondria appears to occur via the electroneutral 2H(+)/Ca(2+)-exchange mechanism, since it can be markedly decreased by cyclosporin A and does not involve lipid peroxidation. When Fe(2+) accumulation is completed, reuptake of released Ca(2+) into mitochondria resumes. Finally, we propose that Fe(2+) either inhibits Ca(2+) entry at the uniporter or is transported by it into the matrix.     

Mitochondrial Ca2+-activated K+ channels more efficiently reduce mitochondrial Ca2+ overload in rat ventricular myocytes

We investigated the role of the mitochondrial ATP-sensitive K(+) (K(ATP)) channel, the mitochondrial big-conductance Ca(2+)-activated K(+) (BK(Ca)) channel, and the mitochondrial permeability transition pore (MPTP) in the ouabain-induced increase of mitochondrial Ca(2+) in native rat ventricular myocytes by loading cells with rhod 2-AM. To overload mitochondrial Ca(2+), we pretreated cells with ouabain before applying mitochondrial K(ATP) or BK(Ca) channel and/or MPTP opener. Ouabain (1 mM) increased the rhod 2-sensitive fluorescence intensity (160 +/- 5.0% of control), which was dramatically decreased to the control level on application of diazoxide and NS-1619 in a dose-dependent manner (half-inhibition concentrations of 78.3 and 7.78 muM for diazoxide and NS-1619, respectively). This effect was reversed by selective inhibition of the mitochondrial K(ATP) channel by 5-hydroxydecanoate, the mitochondrial BK(Ca) channel by paxilline, and the MPTP by cyclosporin A. Although diazoxide did not efficiently reduce mitochondrial Ca(2+) during prolonged exposure to ouabain, NS-1619 reduced mitochondrial Ca(2+). These results suggest that although mitochondrial BK(Ca) and K(ATP) channels contribute to reduction of ouabain-induced mitochondrial Ca(2+) overload, activation of the mitochondrial BK(Ca) channel more efficiently reduces ouabain-induced mitochondrial Ca(2+) overload in our experimental model.     

The mitochondrial Na(+)/Ca(2+) exchanger

Powered by the steep mitochondrial membrane potential Ca(2+) permeates into the mitochondria via the Ca(2+) uniporter and is then extruded by a mitochondrial Na(+)/Ca(2+) exchanger. This mitochondrial Ca(2+) shuttling regulates the rate of ATP production and participates in cellular Ca(2+) signaling. Despite the fact that the exchanger was functionally identified 40 years ago its molecular identity remained a mystery. Early studies on isolated mitochondria and intact cells characterized the functional properties of a mitochondrial Na(+)/Ca(2+) exchanger, and showed that it possess unique functional fingerprints such as Li(+)/Ca(2+) exchange and that it is displaying selective sensitivity to inhibitors. Purification of mitochondria proteins combined with functional reconstitution led to the isolation of a polypeptide candidate of the exchanger but failed to molecularly identify it. A turning point in the search for the exchanger molecule came with the recent cloning of the last member of the Na(+)/Ca(2+) exchanger superfamily termed NCLX (Na(+)/Ca(2+)/Li(+) exchanger). NCLX is localized in the inner mitochondria membrane and its expression is linked to mitochondria Na(+)/Ca(2+) exchange matching the functional fingerprints of the putative mitochondrial Na(+)/Ca(2+) exchanger. Thus NCLX emerges as the long sought mitochondria Na(+)/Ca(2+) exchanger and provide a critical molecular handle to study mitochondrial Ca(2+) signaling and transport. Here we summarize some of the main topics related to the molecular properties of the Na(+)/Ca(2+) exchanger, beginning with the early days of its functional identification, its kinetic properties and regulation, and culminating in its molecular identification.     

Effect of magnesium on calcium-induced depolarisation of mitochondrial transmembrane potential

An effect of magnesium on calcium-induced depolarisation of mitochondrial transmembrane potential (DeltaPsi(m)) was investigated. Depending on the presence of Mg(2+), addition of Ca(2+) to suspension of isolated rat heart mitochondria induced either reversible depolarisation or irreversible collapse of succinate-driven DeltaPsi(m). Irreversible collapse of DeltaPsi(m), observed in the absence of Mg(2+), was insensitive to Ca(2+) chelation, inhibition of Ca(2+) uptake and increased efflux of Ca(2+) from mitochondrial matrix. Based on these data, opening of mPTP in a high-conductance mode is considered to be a major cause of the Ca(2+)-induced irreversible collapse of DeltaPsi(m) in the absence of Mg(2+). Involvement of mPTP in the process of Ca(2+)-induced collapse of DeltaPsi(m) was further supported by protective effect of both CsA and ADP. Reversible collapse of DeltaPsi(m), observed in the presence of Mg(2+), was sensitive to EGTA, ADP; and inhibition of Ca(2+) uptake and increased efflux of Ca(2+) from mitochondrial matrix. This may represent selective induction of a low-conductance permeability pathway. Presented results indicate important role of Mg(2+) in the process of Ca(2+)-induced depolarisation of DeltaPsi(m) mainly through discrimination between low- and high-conductance modes of mPTP. Minor effect of Mg(2+) on Ca(2+)-induced depolarisation of DeltaPsi(m) was observed at the level of stimulation of DeltaPsi(m) generation and inhibition of mitochondrial Ca(2+) uptake.