Characterization of Mg2+ inhibition of mitochondrial Ca2+ uptake by a mechanistic model of mitochondrial Ca2+ uniporter

Ca(2+) is an important regulatory ion and alteration of mitochondrial Ca(2+) homeostasis can lead to cellular dysfunction and apoptosis. Ca(2+) is transported into respiring mitochondria via the Ca(2+) uniporter, which is known to be inhibited by Mg(2+). This uniporter-mediated mitochondrial Ca(2+) transport is also shown to be influenced by inorganic phosphate (Pi). Despite a large number of experimental studies, the kinetic mechanisms associated with the Mg(2+) inhibition and Pi regulation of the uniporter function are not well established. To gain a quantitative understanding of the effects of Mg(2+) and Pi on the uniporter function, we developed here a mathematical model based on known kinetic properties of the uniporter and presumed Mg(2+) 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 Ca(2+) uptake. The model also appropriately depicts the inhibitory effect of Mg(2+) on the uniporter function, in which Ca(2+) uptake is hyperbolic in the absence of Mg(2+) and sigmoid in the presence of Mg(2+). The model suggests a mixed-type inhibition mechanism for Mg(2+) inhibition of the uniporter function. This model is critical for building mechanistic models of mitochondrial bioenergetics and Ca(2+) handling to understand the mechanisms by which Ca(2+) mediates signaling pathways and modulates energy metabolism.     

The endoplasmic reticulum gateway to apoptosis by Bcl-X(L) modulation of the InsP3R

Members of the Bcl-2 protein family modulate outer mitochondrial membrane permeability to control apoptosis. However, these proteins also localize to the endoplasmic reticulum (ER), the functional significance of which is controversial. Here we provide evidence that anti-apoptotic Bcl-2 proteins regulate the inositol 1,4,5-trisphosphate receptor (InsP(3)R) ER Ca(2+) release channel resulting in increased cellular apoptotic resistance and enhanced mitochondrial bioenergetics. Anti-apoptotic Bcl-X(L) interacts with the carboxyl terminus of the InsP(3)R and sensitizes single InsP(3)R channels in ER membranes to low [InsP(3)], enhancing Ca(2+) and InsP(3)-dependent regulation of channel activity in vitro and in vivo, reducing ER Ca(2+) content and stimulating mitochondrial energetics. The pro-apoptotic proteins Bax and tBid antagonize this effect by blocking the biochemical interaction of Bcl-X(L) with the InsP(3)R. These data support a novel model in which Bcl-X(L) is a direct effector of the InsP(3)R, increasing its sensitivity to InsP(3) and enabling ER Ca(2+) release to be more sensitively coupled to extracellular signals. As a consequence, cells are protected against apoptosis by a more sensitive and dynamic coupling of ER to mitochondria through Ca(2+)-dependent signal transduction that enhances cellular bioenergetics and preserves survival.     

An integrated model of cardiac mitochondrial energy metabolism and calcium dynamics

We present an integrated thermokinetic model describing control of cardiac mitochondrial bioenergetics. The model describes the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, and mitochondrial Ca(2+) handling. The kinetic component of the model includes effectors of the TCA cycle enzymes regulating production of NADH and FADH(2), which in turn are used by the electron transport chain to establish a proton motive force (Delta mu(H)), driving the F(1)F(0)-ATPase. In addition, mitochondrial matrix Ca(2+), determined by Ca(2+) uniporter and Na(+)/Ca(2+) exchanger activities, regulates activity of the TCA cycle enzymes isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase. The model is described by twelve ordinary differential equations for the time rate of change of mitochondrial membrane potential (Delta Psi(m)), and matrix concentrations of Ca(2+), NADH, ADP, and TCA cycle intermediates. The model is used to predict the response of mitochondria to changes in substrate delivery, metabolic inhibition, the rate of adenine nucleotide exchange, and Ca(2+). The model is able to reproduce, qualitatively and semiquantitatively, experimental data concerning mitochondrial bioenergetics, Ca(2+) dynamics, and respiratory control. Significant increases in oxygen consumption (V(O(2))), proton efflux, NADH, and ATP synthesis, in response to an increase in cytoplasmic Ca(2+), are obtained when the Ca(2+)-sensitive dehydrogenases are the main rate-controlling steps of respiratory flux. These responses diminished when control is shifted downstream (e.g., the respiratory chain or adenine nucleotide translocator). The time-dependent behavior of the model, under conditions simulating an increase in workload, closely reproduces experimentally observed mitochondrial NADH dynamics in heart trabeculae subjected to changes in pacing frequency. The steady-state and time-dependent behavior of the model support the hypothesis that mitochondrial matrix Ca(2+) plays an important role in matching energy supply with demand in cardiac myocytes.     

Role of proton gradients and vacuola H(+)-ATPases in the refilling of intracellular calcium stores in exocrine cells

Numerous hormones and neurotransmitters activate cells by increasing cytosolic calcium concentration ([Ca(2+)](i)), a key regulatory factor for many cellular processes. A pivotal feature of these Ca(2+) signals is the release of Ca(2+) from intracellular stores, which is followed by activation of extracellular calcium influx, allowing refilling of the stores by SERCA pumps associated with the endoplasmic reticulum. Although the mechanisms of calcium release and calcium influx have been extensively studied, the biology of the Ca(2+) stores is poorly understood. The presence of heterogeneous calcium pools in cells has been previously reported [1] [2] [3]. Although recent technical improvements have confirmed this heterogeneity [4], knowledge about the mechanisms underlying Ca(2+) transport within the stores is very scarce and rather speculative. A recent study in polarized exocrine cells [5] has revealed the existence of Ca(2+) tunneling from basolateral stores to luminal pools, where Ca(2+) is initially released upon cell activation. Here, we present evidence that, during stimulation, Ca(2+) transported into basolateral stores by SERCA pumps is conveyed toward the luminal pools driven by proton gradients generated by vacuolar H(+)-ATPases. This finding unveils a new aspect of the machinery of Ca(2+) stores.     

Mitochondrial calcium transport: mechanisms and functions

Ca(2+)transport across the mitochondrial inner membrane is facilitated by transporters having four distinct sets of characteristics as well as through the Ca(2+)-induced mitochondrial permeability transition pore (PTP). There are two modes of inward transport, referred to as the Ca(2+)uniporter and the rapid mode or RaM. There are also two distinct mechanisms mediating outward transport, which are not associated with the PTP, referred to as the Na(+)-dependent and the Na(+)-independent Ca(2+)efflux mechanisms. Several important functions have been proposed for these mechanisms, including control of the metabolic rate for cellular energy (ATP) production, modulation of the amplitude and shape of cytosolic Ca(2+)transients, and induction of apoptosis through release of cytochrome c from the mitochondrial inter membrane space into the cytosolic space.The goals of this review are to survey the literature describing the characteristics of the mechanisms of mitochondrial Ca(2+)transport and their proposed physiological functions, emphasizing the more recent contributions, and to consider how the observed characteristics of the mitochondrial Ca(2+)transport mechanisms affect our understanding of their functions.     

Ca(2+) signalling in mitochondria: mechanism and role in physiology and pathology

Over recent years, a renewed interest in mitochondria in the field of Ca(2+) signalling has highlighted their central role in regulating important physiological and pathological events in animal cells. Mitochondria take up calcium through an uptake pathway that, due to its low-Ca(2+) affinity, demands high local calcium concentrations to work. In different cell systems high-Ca(2+) concentration microdomains are generated, upon cell stimulation, in proximity of either plasma membrane or sarco/endoplasmic reticulum Ca(2+) channels. Mitochondrial Ca(2+) accumulation has a dual role, an universal one, which consists in satisfying energy demands by increasing the ATP production through the activation of mitochondrial enzymes, and a cell type specific one, which, through the modulation of the spatio-temporal dynamics of calcium signals, contributes to modulate specific cell functions. Recent work has revealed the central role of mitochondria dysfunction in determining both necrotic and apoptotic cell death. Evidence is also accumulating that suggests that alterations in mitochondrial function may act as predisposing factors in the pathogenesis of a number of neurodegenerative disorders. These include inherited disorders of the mitochondrial genome in which a defect in mitochondrial calcium accumulation has been shown to correlate with a defect in ATP production, thus suggesting a possible involvement of mitochondrial Ca(2+) dysfunction also for this group of diseases. This review analyses recent developments in the area of mitochondrial Ca(2+) signalling and attempts to summarise cell physiology and cell pathology aspects of the mitochondrial Ca(2+) transport machinery.     

Identification of a ryanodine receptor in rat heart mitochondria

Recent studies have shown that, in a wide variety of cells, mitochondria respond dynamically to physiological changes in cytosolic Ca(2+) concentrations ([Ca(2+)](c)). Mitochondrial Ca(2+) uptake occurs via a ruthenium red-sensitive calcium uniporter and a rapid mode of Ca(2+) uptake. Surprisingly, the molecular identity of these Ca(2+) transport proteins is still unknown. Using electron microscopy and Western blotting, we identified a ryanodine receptor in the inner mitochondrial membrane with a molecular mass of approximately 600 kDa in mitochondria isolated from the rat heart. [(3)H]Ryanodine binds to this mitochondrial ryanodine receptor with high affinity. This binding is modulated by Ca(2+) but not caffeine and is inhibited by Mg(2+) and ruthenium red in the assay medium. In the presence of ryanodine, Ca(2+) uptake into isolated heart mitochondria is suppressed. In addition, ryanodine inhibited mitochondrial swelling induced by Ca(2+) overload. This swelling effect was not observed when Ca(2+) was applied to the cytosolic fraction containing sarcoplasmic reticulum. These results are the first to identify a mitochondrial Ca(2+) transport protein that has characteristics similar to the ryanodine receptor. This mitochondrial ryanodine receptor is likely to play an essential role in the dynamic uptake of Ca(2+) into mitochondria during Ca(2+) oscillations.     

α-Synuclein controls mitochondrial calcium homeostasis by enhancing endoplasmic reticulum-mitochondria interactions

α-Synuclein has a central role in Parkinson disease, but its physiological function and the mechanism leading to neuronal degeneration remain unknown. Because recent studies have highlighted a role for α-synuclein in regulating mitochondrial morphology and autophagic clearance, we investigated the effect of α-synuclein in HeLa cells on mitochondrial signaling properties focusing on Ca(2+) homeostasis, which controls essential bioenergetic functions. By using organelle-targeted Ca(2+)-sensitive aequorin probes, we demonstrated that α-synuclein positively affects Ca(2+) transfer from the endoplasmic reticulum to the mitochondria, augmenting the mitochondrial Ca(2+) transients elicited by agonists that induce endoplasmic reticulum Ca(2+) release. This effect is not dependent on the intrinsic Ca(2+) uptake capacity of mitochondria, as measured in permeabilized cells, but correlates with an increase in the number of endoplasmic reticulum-mitochondria interactions. This action specifically requires the presence of the C-terminal α-synuclein domain. Conversely, α-synuclein siRNA silencing markedly reduces mitochondrial Ca(2+) uptake, causing profound alterations in organelle morphology. The enhanced accumulation of α-synuclein into the cells causes the redistribution of α-synuclein to localized foci and, similarly to the silencing of α-synuclein, reduces the ability of mitochondria to accumulate Ca(2+). The absence of efficient Ca(2+) transfer from endoplasmic reticulum to mitochondria results in augmented autophagy that, in the long range, could compromise cellular bioenergetics. Overall, these findings demonstrate a key role for α-synuclein in the regulation of mitochondrial homeostasis in physiological conditions. Elevated α-synuclein expression and/or eventually alteration of the aggregation properties cause the redistribution of the protein within the cell and the loss of modulation on mitochondrial function.     

Old players in a new role: mitochondria-associated membranes,VDAC, and ryanodine receptors as contributors to calcium signal propagation from endoplasmic reticulum to the mitochondria

In many cell types, IP(3) and ryanodine receptor (IP(3)R/RyR)-mediated Ca(2+) mobilization from the sarcoendoplasmic reticulum (ER/SR) results in an elevation of mitochondrial matrix [Ca(2+)]. Although delivery of the released Ca(2+) to the mitochondria has been established as a fundamental signaling process, the molecular mechanism underlying mitochondrial Ca(2+) uptake remains a challenge for future studies. The Ca(2+) uptake can be divided into the following three steps: (1) Ca(2+) movement from the IP(3)R/RyR to the outer mitochondrial membrane (OMM); (2) Ca(2+) transport through the OMM; and (3) Ca(2+) transport through the inner mitochondrial membrane (IMM). Evidence has been presented that Ca(2+) delivery to the OMM is facilitated by a local coupling between closely apposed regions of the ER/SR and mitochondria. Recent studies of the dynamic changes in mitochondrial morphology and visualization of the subcellular pattern of the calcium signal provide important clues to the organization of the ER/SR-mitochondrial interface. Interestingly, key steps of phospholipid synthesis and transfer to the mitochondria have also been confined to subdomains of the ER tightly associated with the mitochondria, referred as mitochondria-associated membranes (MAMs). Through the OMM, the voltage-dependent anion channels (VDAC, porin) have been thought to permit free passage of ions and other small molecules. However, recent studies suggest that the VDAC may represent a regulated step in Ca(2+) transport from IP(3)R/RyR to the IMM. A novel proposal regarding the IMM Ca(2+) uptake site is a mitochondrial RyR that would mediate rapid Ca(2+) uptake by mitochondria in excitable cells. An overview of the progress in these directions is described in the present paper.     

Mitochondrial Ca(2+) signals in autophagy

Macroautophagy (autophagy) is a lysosomal degradation pathway that is conserved from yeast to humans that plays an important role in recycling cellular constituents in all cells. A number of protein complexes and signaling pathways impinge on the regulation of autophagy, with the mammalian target of rapamycin (mTOR) as the central player in the canonical pathway. Cytoplasmic Ca(2+) signaling also regulates autophagy, with both activating and inhibitory effects, mediated by the canonical as well as non-canonical pathways. Here we review this regulation, with a focus on the role of an mTOR-independent pathway that involves the inositol trisphosphate receptor (InsP(3)R) Ca(2+) release channel and Ca(2+) signaling to mitochondria. Constitutive InsP(3)R Ca(2+) transfer to mitochondria is required for autophagy suppression in cells in nutrient-replete media. In its absence, cells become metabolically compromised due to insufficient production of reducing equivalents to support oxidative phosphorylation. Absence of this Ca(2+) transfer to mitochondria results in activation of AMPK, which activates mTOR-independent pro-survival autophagy. Constitutive InsP(3)R Ca(2+) release to mitochondria is an essential cellular process that is required for efficient mitochondrial respiration, maintenance of normal cell bioenergetics and suppression of autophagy.     

Mitochondrial Ca(2+) and neurodegeneration

Mitochondria are essential for ensuring numerous fundamental physiological processes such as cellular energy, redox balance, modulation of Ca(2+) signaling and important biosynthetic pathways. They also govern the cell fate by participating in the apoptosis pathway. The mitochondrial shape, volume, number and distribution within the cells are strictly controlled. The regulation of these parameters has an impact on mitochondrial function, especially in the central nervous system, where trafficking of mitochondria is critical to their strategic intracellular distribution, presumably according to local energy demands. Thus, the maintenance of a healthy mitochondrial population is essential to avoid the impairment of the processes they regulate: for this purpose, cells have developed mechanisms involving a complex system of quality control to remove damaged mitochondria, or to renew them. Defects of these processes impair mitochondrial function and lead to disordered cell function, i.e., to a disease condition. Given the standard role of mitochondria in all cells, it might be expected that their dysfunction would give rise to similar defects in all tissues. However, damaged mitochondrial function has pleiotropic effects in multicellular organisms, resulting in diverse pathological conditions, ranging from cardiac and brain ischemia, to skeletal muscle myopathies to neurodegenerative diseases. In this review, we will focus on the relationship between mitochondrial (and cellular) derangements and Ca(2+) dysregulation in neurodegenerative diseases, emphasizing the evidence obtained in genetic models. Common patterns, that recognize the derangement of Ca(2+) and energy control as a causative factor, have been identified: advances in the understanding of the molecular regulation of Ca(2+) homeostasis, and on the ways in which it could become perturbed in neurological disorders, may lead to the development of therapeutic strategies that modulate neuronal Ca(2+) signaling.     

Functional coupling between ryanodine receptors, mitochondria and Ca(2+) ATPases in rat submandibular acinar cells

Agonist stimulation of exocrine cells leads to the generation of intracellular Ca(2+) signals driven by inositol 1,4,5-trisphosphate receptors (IP(3)Rs) that rapidly become global due to propagation throughout the cell. In many types of excitable cells the intracellular Ca(2+) signal is propagated by a mechanism of Ca(2+)-induced Ca(2+) release (CICR), mediated by ryanodine receptors (RyRs). Expression of RyRs in salivary gland cells has been demonstrated immunocytochemically although their functional role is not clear. We used microfluorimetry to measure Ca(2+) signals in the cytoplasm, in the endoplasmic reticulum (ER) and in mitochondria. In permeabilized acinar cells caffeine induced a dose-dependent, transient decrease of Ca(2+) concentration in the endoplasmic reticulum ([Ca(2+)](ER)). This decrease was inhibited by ryanodine but was insensitive to heparin. Application of caffeine, however, did not elevate cytosolic Ca(2+) concentration ([Ca(2+)](i)) suggesting fast local buffering of Ca(2+) released through RyRs. Indeed, activation of RyRs produced a robust mitochondrial Ca(2+) transient that was prevented by addition of Ca(2+) chelator BAPTA but not EGTA. When mitochondrial Ca(2+) uptake was blocked, activation of RyRs evoked only a non-transient increase in [Ca(2+)](i) and substantially smaller Ca(2+) release from the ER. Upon simultaneous inhibition of mitochondrial Ca(2+) uptake and either plasmalemmal or ER Ca(2+) ATPase, activation of RyRs caused a transient rise in [Ca(2+)](i). Collectively, our data suggest that Ca(2+) released through RyRs is mostly "tunnelled" to mitochondria, while Ca(2+) ATPases are responsible for the fast initial sequestration of Ca(2+). Ca(2+) uptake by mitochondria is critical for maintaining continuous CICR. A complex interplay between RyRs, mitochondria and Ca(2+) ATPases is accomplished through strategic positioning of mitochondria close to both Ca(2+) release sites in the ER and Ca(2+) pumping sites of the plasmalemma and the ER.     

Calcium and mitochondria: mechanisms and functions of a troubled relationship

Mitochondria promptly respond to Ca(2+)-mediated cell stimulations with a rapid accumulation of the cation into the matrix. In this article, we review (i) the basic principles of mitochondrial Ca(2+) transport, (ii) the physiological/pathological role of mitochondrial Ca(2+) uptake, (iii) the regulatory mechanisms that may operate in vivo, and (iv) the new targeted Ca(2+) probes that allowed the "rediscovery" of these organelles in calcium signalling.     

Characterization of the intracellular Ca(2+) pools involved in the calcium homeostasis in Herpetomonas sp. promastigotes

Trypanosomatids of the genus Herpetomonas comprises monoxenic parasites of insects that present pro- and opisthomastigotes forms in their life cycles. In this study, we investigated the Ca(2+) transport and the mitochondrial bioenergetic of digitonin-permeabilized Herpetomonas sp. promastigotes. The response of promastigotes mitochondrial membrane potential to ADP, oligomycin, Ca(2+), and antimycin A indicates that these mitochondria behave similarly to vertebrate and Trypanosoma cruzi mitochondria regarding the properties of their electrochemical proton gradient. Ca(2+) transport by permeabilized cells appears to be performed mainly by the mitochondria. Unlike T. cruzi, it was not possible to observe Ca(2+) release from Herpetomonas sp. mitochondria, probably due to the simultaneous Ca(2+) uptake by the endoplasmic reticulum. In addition, a vanadate-sensitive Ca(2+) transport system, attributed to the endoplasmic reticulum, was also detected. Nigericin (1 microM), FCCP (1 microM), or bafilomycin A(1) (5 microM) had no effect on the vanadate-sensitive Ca(2+) transport. These data suggest the absence of a Ca(2+) transport mediated by a Ca(2+)/H(+) antiport. No evidence of a third Ca(2+) compartment with the characteristics of the acidocalcisomes described by A. E. Vercesi et al. (1994, Biochem. J. 304, 227-233) was observed. Thapsigargin and IP(3) were not able to affect the vanadate-sensitive Ca(2+) transport. Ruthenium red was able to inhibit the Ca(2+) uniport of mitochondria, inducing a slow mitochondrial Ca(2+) efflux, compatible with the presence of a Ca(2+)/H(+) antiport. Moreover, this efflux was not stimulated by the addition of NaCl, which suggests the absence of a Ca(2+)/Na(+) antiport in mitochondria.     

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.     

Ca2+ signaling in the regulation of dendritic cell functions

Dendritic cells (DCs) are highly versatile antigen-presenting cells critically involved in both innate and adaptive immunity as well as maintenance of self-tolerance. DC function is governed by Ca(2+) signaling, which directs the DC responses to diverse antigens, including Toll-like receptor ligands, intact bacteria, and microbial toxins. Ca(2+)-sensitive DC functions include DC activation, maturation, migration, and formation of immunological synapses with T cells. Moreover, alterations of cytosolic Ca(2+) trigger immune suppression or switch off DC activity. Ca(2+) signals are generated by the orchestration of Ca(2+) transport processes across plasma, endoplasmic reticulum, and inner mitochondrial membrane. These processes include active pumping of Ca(2+), Ca(2+)/Na(+) antiport, and electrodiffusion through Ca(2+)-permeable channels or uniporters. Ca(2+) channels in the plasma membrane such as Ca(2+) release-activated Ca(2+) or L-type Ca(2+) channels are tightly regulated by the membrane potential which in turn depends on the activity of voltage-gated K(+) or Ca(2+)-activated nonselective cation channels. The rapidly growing knowledge on the function and regulation of these membrane transport proteins provides novel insight into pathophysiological mechanisms underlying dysfunction of the immune system and opens novel therapeutic opportunity to favorably influence the function of the immune system.     

High- and low-calcium-dependent mechanisms of mitochondrial calcium signalling

The Ca(2+) coupling between endoplasmic reticulum (ER) and mitochondria is central to multiple cell survival and cell death mechanisms. Cytoplasmic [Ca(2+)] ([Ca(2+)](c)) spikes and oscillations produced by ER Ca(2+) release are effectively delivered to the mitochondria. Propagation of [Ca(2+)](c) signals to the mitochondria requires the passage of Ca(2+) across three membranes, namely the ER membrane, the outer mitochondrial membrane (OMM) and the inner mitochondrial membrane (IMM). Strategic positioning of the mitochondria by cytoskeletal transport and interorganellar tethers provides a means to promote the local transfer of Ca(2+) between the ER membrane and OMM. In this setting, even >100 microM [Ca(2+)] may be attained to activate the low affinity mitochondrial Ca(2+) uptake. However, a mitochondrial [Ca(2+)] rise has also been documented during submicromolar [Ca(2+)](c) elevations. Evidence has been emerging that Ca(2+) exerts allosteric control on the Ca(2+) transport sites at each membrane, providing mechanisms that may facilitate the Ca(2+) delivery to the mitochondria. Here we discuss the fundamental mechanisms of ER and mitochondrial Ca(2+) transport, particularly the control of their activity by Ca(2+) and evaluate both high- and low-[Ca(2+)]-activated mitochondrial calcium signals in the context of cell physiology.     

Species dependence of mitochondrial calcium transients during excitation-contraction coupling in isolated cardiomyocytes.

Whether mitochondrial Ca(2+) transport is rapid enough to respond to changes in cytosolic [Ca(2+)] ([Ca(2+)](c)) which occur during excitation-contraction coupling in the heart is controversial; different results wereobtained with different techniques and different species. In this study mitochondrial [Ca(2+)] ([Ca(2+)](m)) was measured in indo-1/AM-loaded myocytes from rat and guinea-pig hearts where the cytosolic indo-1 had been removed by extended incubation of cells at 37 degrees C ("heat treatment"). The mitochondrial origin of the remaining fluorescence was confirmed by sensitivity of the indo-1 signal to ruthenium red. In resting rat myocytes, [Ca(2+)](m) was lower than [Ca(2+)](c), whereas in guinea-pig cells [Ca(2+)](m) was higher than [Ca(2+)](c). Upon electrical stimulation of cells, no change occurred in [Ca(2+)](m) in rat myocytes. However, in guinea-pig cells mitochondrial Ca(2+) transients were clearly visible with a mean indo-1 ratio amplitude of 0.153 +/- 0.2 (n = 20), compared with 0.306 +/- 0.02 (n = 25), p < 0.001, prior to heat treatment. These observations suggest significant differences in mitochondrial Ca(2+) transport in cardiomyocytes from different species.