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.     

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.     

Mitochondrial Ca(2+)-induced Ca(2+) release mediated by the Ca(2+) uniporter

We have reported that a population of chromaffin cell mitochondria takes up large amounts of Ca(2+) during cell stimulation. The present study focuses on the pathways for mitochondrial Ca(2+) efflux. Treatment with protonophores before cell stimulation abolished mitochondrial Ca(2+) uptake and increased the cytosolic [Ca(2+)] ([Ca(2+)](c)) peak induced by the stimulus. Instead, when protonophores were added after cell stimulation, they did not modify [Ca(2+)](c) kinetics and inhibited Ca(2+) release from Ca(2+)-loaded mitochondria. This effect was due to inhibition of mitochondrial Na(+)/Ca(2+) exchange, because blocking this system with CGP37157 produced no further effect. Increasing extramitochondrial [Ca(2+)](c) triggered fast Ca(2+) release from these depolarized Ca(2+)-loaded mitochondria, both in intact or permeabilized cells. These effects of protonophores were mimicked by valinomycin, but not by nigericin. The observed mitochondrial Ca(2+)-induced Ca(2+) release response was insensitive to cyclosporin A and CGP37157 but fully blocked by ruthenium red, suggesting that it may be mediated by reversal of the Ca(2+) uniporter. This novel kind of mitochondrial Ca(2+)-induced Ca(2+) release might contribute to Ca(2+) clearance from mitochondria that become depolarized during Ca(2+) overload.     

tcBid promotes Ca(2+) signal propagation to the mitochondria: control of Ca(2+) permeation through the outer mitochondrial membrane

Calcium spikes established by IP(3) receptor-mediated Ca(2+) release from the endoplasmic reticulum (ER) are transmitted effectively to the mitochondria, utilizing local Ca(2+) interactions between closely associated subdomains of the ER and mitochondria. Since the outer mitochondrial membrane (OMM) has been thought to be freely permeable to Ca(2+), investigations have focused on IP(3)-driven Ca(2+) transport through the inner mitochondrial membrane (IMM). Here we demonstrate that selective permeabilization of the OMM by tcBid, a proapoptotic protein, results in an increase in the magnitude of the IP(3)-induced mitochondrial [Ca(2+)] signal. This effect of tcBid was due to promotion of activation of Ca(2+) uptake sites in the IMM and, in turn, to facilitation of mitochondrial Ca(2+) uptake. In contrast, tcBid failed to control the delivery of sustained and global Ca(2+) signals to the mitochondria. Thus, our data support a novel model that Ca(2+) permeability of the OMM at the ER- mitochondrial interface is an important determinant of local Ca(2+) signalling. Facilitation of Ca(2+) delivery to the mitochondria by tcBid may also support recruitment of mitochondria to the cell death machinery.     

Mitochondrial Ca(2+) uptake depends on the spatial and temporal profile of cytosolic Ca(2+) signals

Using confocal imaging of Rhod-2-loaded HeLa cells, we examined the ability of mitochondria to sequester Ca(2+) signals arising from different sources. Mitochondrial Ca(2+) (Ca(2+)mit) uptake was stimulated by inositol 1,4,5-trisphosphate (InsP(3))-evoked Ca(2+) release, capacitative Ca(2+) entry, and Ca(2+) leaking from the endoplasmic reticulum. For each Ca(2+) source, the relationship between cytosolic Ca(2+) (Ca(2+)cyt) concentration and Ca(2+)mit was complex. With Ca(2+)cyt < 300 nm, a slow and persistent Ca(2+)mit uptake was observed. If Ca(2+)cyt increased above approximately 400 nm, Ca(2+)mit uptake accelerated sharply. For equivalent Ca(2+)cyt increases, the rate of Ca(2+)mit rise was greater with InsP(3)-evoked Ca(2+) signals than any other source. Spatial variation of the Ca(2+)mit response was observed within individual cells. Both the fraction of responsive mitochondria and the amplitude of the Ca(2+)mit response were graded in direct proportion to stimulus concentration. Trains of repetitive Ca(2+) oscillations did not maintain elevated Ca(2+)mit levels. Only low frequency Ca(2+) transients (<1/15 min) evoked repetitive Ca(2+)mit signals. Our data indicate that there is a lag between Ca(2+)cyt and Ca(2+)mit increases but that mitochondria will accumulate calcium when it is elevated over basal levels regardless of its source. Furthermore, in addition to the characteristics of Ca(2+) signals, Ca(2+) uniporter desensitization and proximity of mitochondria to InsP(3) receptors modulate mitochondrial Ca(2+) responses.     

Mitochondria couple cellular Ca(2+) signal transduction

It has been shown that mitochondria not only control their own Ca(2+) concentration ([Ca(2+)]), but also exert an influence over Ca(2+) signaling of the entire cell, including the endoplasmic reticulum or the sarcoplasmic reticulum, the plasma membrane, and the nucleus. That is to say, mitochondria couple cellular metabolic state with Ca(2+) transport processes. This review focuses on the ways in which the mitochondrial Ca(2+) handling system provides integrity and modulation for the cell to cope with the complex actions throughout its life cycle, enumerates some indeterminate aspects about it, and finally, prospects directions of future research.     

Inositol 1,4,5-trisphosphate directs Ca(2+) flow between mitochondria and the Endoplasmic/Sarcoplasmic reticulum: a role in regulating cardiac autonomic Ca(2+) spiking

The signaling role of the Ca(2+) releaser inositol 1,4, 5-trisphosphate (IP(3)) has been associated with diverse cell functions. Yet, the physiological significance of IP(3) in tissues that feature a ryanodine-sensitive sarcoplasmic reticulum has remained elusive. IP(3) generated by photolysis of caged IP(3) or by purinergic activation of phospholipase Cgamma slowed down or abolished autonomic Ca(2+) spiking in neonatal rat cardiomyocytes. Microinjection of heparin, blocking dominant-negative fusion protein, or anti-phospholipase Cgamma antibody prevented the IP(3)-mediated purinergic effect. IP(3) triggered a ryanodine- and caffeine-insensitive Ca(2+) release restricted to the perinuclear region. In cells loaded with Rhod2 or expressing a mitochondria-targeted cameleon and TMRM to monitor mitochondrial Ca(2+) and potential, IP(3) induced transient Ca(2+) loading and depolarization of the organelles. These mitochondrial changes were associated with Ca(2+) depletion of the sarcoplasmic reticulum and preceded the arrest of cellular Ca(2+) spiking. Thus, IP(3) acting within a restricted cellular region regulates the dynamic of calcium flow between mitochondria and the endoplasmic/sarcoplasmic reticulum. We have thus uncovered a novel role for IP(3) in excitable cells, the regulation of cardiac autonomic activity.     

Uncoupling protein 3 adjusts mitochondrial Ca(2+) uptake to high and low Ca(2+) signals

Uncoupling proteins 2 and 3 (UCP2/3) are essential for mitochondrial Ca(2+) uptake but both proteins exhibit distinct activities in regard to the source and mode of Ca(2+) mobilization. In the present work, structural determinants of their contribution to mitochondrial Ca(2+) uptake were explored. Previous findings indicate the importance of the intermembrane loop 2 (IML2) for the contribution of UCP2/3. Thus, the IML2 of UCP2/3 was substituted by that of UCP1. These chimeras had no activity in mitochondrial uptake of intracellularly released Ca(2+), while they mimicked the wild-type proteins by potentiating mitochondrial sequestration of entering Ca(2+). Alignment of the IML2 sequences revealed that UCP1, UCP2 and UCP3 share a basic amino acid in positions 163, 164 and 167, while only UCP2 and UCP3 contain a second basic residue in positions 168 and 171, respectively. Accordingly, mutants of UCP3 in positions 167 and 171/172 were made. In permeabilized cells, these mutants exhibited distinct Ca(2+) sensitivities in regard to mitochondrial Ca(2+) sequestration. In intact cells, these mutants established different activities in mitochondrial uptake of either intracellularly released (UCP3(R171,E172)) or entering (UCP3(R167)) Ca(2+). Our data demonstrate that distinct sites in the IML2 of UCP3 effect mitochondrial uptake of high and low Ca(2+) signals.     

In vivo monitoring of Ca(2+) uptake into mitochondria of mouse skeletal muscle during contraction

Although the importance of mitochondria in patho-physiology has become increasingly evident, it remains unclear whether these organelles play a role in Ca(2+) handling by skeletal muscle. This undefined situation is mainly due to technical limitations in measuring Ca(2+) transients reliably during the contraction-relaxation cycle. Using two-photon microscopy and genetically expressed "cameleon" Ca(2+) sensors, we developed a robust system that enables the measurement of both cytoplasmic and mitochondrial Ca(2+) transients in vivo. We show here for the first time that, in vivo and under highly physiological conditions, mitochondria in mammalian skeletal muscle take up Ca(2+) during contraction induced by motor nerve stimulation and rapidly release it during relaxation. The mitochondrial Ca(2+) increase is delayed by a few milliseconds compared with the cytosolic Ca(2+) rise and occurs both during a single twitch and upon tetanic contraction.     

Ca(2+) homeostasis during mitochondrial fragmentation and perinuclear clustering induced by hFis1

Mitochondria modulate Ca(2+) signals by taking up, buffering, and releasing Ca(2+) at key locations near Ca(2+) release or influx channels. The role of such local interactions between channels and organelles is difficult to establish in living cells because mitochondria form an interconnected network constantly remodeled by coordinated fusion and fission reactions. To study the effect of a controlled disruption of the mitochondrial network on Ca(2+) homeostasis, we took advantage of hFis1, a protein that promotes mitochondrial fission by recruiting the dynamin-related protein, Drp1. hFis1 expression in HeLa cells induced a rapid and complete fragmentation of mitochondria, which redistributed away from the plasma membrane and clustered around the nucleus. Despite the dramatic morphological alteration, hFis1-fragmented mitochondria maintained a normal transmembrane potential and pH and took up normally the Ca(2+) released from intracellular stores upon agonist stimulation, as measured with a targeted ratiometric pericam probe. In contrast, hFis1-fragmented mitochondria took up more slowly the Ca(2+) entering across plasma membrane channels, because the Ca(2+) ions reaching mitochondria propagated faster and in a more coordinated manner in interconnected than in fragmented mitochondria. In parallel cytosolic fura-2 measurements, the capacitative Ca(2+) entry (CCE) elicited by store depletion was only marginally reduced by hFis1 expression. Regardless of mitochondria shape and location, disruption of mitochondrial potential with uncouplers or oligomycin/rotenone reduced CCE by approximately 35%. These observations indicate that close contact to Ca(2+) influx channels is not required for CCE modulation and that the formation of a mitochondrial network facilitates Ca(2+) propagation within interconnected mitochondria.     

Physical coupling supports the local Ca2+ transfer between sarcoplasmic reticulum subdomains and the mitochondria in heart muscle

In many cell types, transfer of Ca(2+) released via ryanodine receptors (RyR) to the mitochondrial matrix is locally supported by high [Ca(2+)] microdomains at close contacts between the sarcoplasmic reticulum (SR) and mitochondria. Here we studied whether the close contacts were secured via direct physical coupling in cardiac muscle using isolated rat heart mitochondria (RHMs). "Immuno-organelle chemistry" revealed RyR2 and calsequestrin-positive SR particles associated with mitochondria in both crude and Percoll-purified "heavy" mitochondrial fractions (cRHM and pRHM), to a smaller extent in the latter one. Mitochondria-associated vesicles were also visualized by electron microscopy in the RHMs. Western blot analysis detected greatly reduced presence of SR markers (calsequestrin, SERCA2a, and phospholamban) in pRHM, suggesting that the mitochondria-associated particles represented a small subfraction of the SR. Fluorescence calcium imaging in rhod2-loaded cRHM revealed mitochondrial matrix [Ca(2+)] ([Ca(2+)](m)) responses to caffeine-induced Ca(2+) release that were prevented when thapsigargin was added to predeplete the SR or by mitochondrial Ca(2+) uptake inhibitors. Importantly, caffeine failed to increase [Ca(2+)] in the large volume of the incubation medium, suggesting that local Ca(2+) transfer between the SR particles and mitochondria mediated the [Ca(2+)](m) signal. Despite the substantially reduced SR presence, pRHM still displayed a caffeine-induced [Ca(2+)](m) rise comparable with the one recorded in cRHM. Thus, a relatively small fraction of the total SR is physically coupled and transfers Ca(2+) locally to the mitochondria in cardiac muscle. The transferred Ca(2+) stimulates dehydrogenase activity and affects mitochondrial membrane permeabilization, indicating the broad significance of the physical coupling in mitochondrial function.     

Modulation of mitochondrial Ca(2+) homeostasis by Bcl-2

We have investigated the role of mitochondrial Ca(2+) (Ca(m)) homeostasis in cell survival. Disruption of Ca(m) homeostasis via depletion of the mitochondrial Ca(2+) store was the earliest event that occurred during staurosporine-induced apoptosis in neuroblastoma cells (SH-SY5Y). The decrease of Ca(m) preceded activation of the caspase cascade and DNA fragmentation. Overexpression of the anti-apoptosis protein Bcl-2 led to increased Ca(m) load, increased mitochondrial membrane potential (DeltaPsi(m)), and inhibition of staurosporine-induced apoptosis. On the other hand, ectopic expression of the pro-apoptotic protein Bik led to decreased Ca(m) load and decreased DeltaPsi(m). Inhibition of calcium uptake into mitochondria by ruthenium red induced a dose-dependent apoptosis as determined by nuclear staining and DNA ladder assay. Similarly, reducing the Ca(m) load by lowering the extracellular calcium concentration also led to apoptosis. We suggest that the anti-apoptotic effect of Bcl-2 is related to its ability to maintain a threshold level of Ca(m) and DeltaPsi(m) while the pro-apoptotic protein Bik has the opposite effect. Furthermore, both ER and mitochondrial Ca(2+) stores are important, and the depletion of either one will result in apoptosis. Thus, our results, for the first time, provide evidence that the maintenance of Ca(m) homeostasis is essential for cell survival.     

Effects of polyamines on mitochondrial Ca(2+) transport

Mammalian mitochondria are able to enhance Ca(2+) accumulation in the presence of polyamines by activating the saturable systems of Ca(2+) inward transport and buffering extramitochondrial Ca(2+) concentrations to levels similar to those in the cytosol of resting cells. This effect renders them responsive to regulate free Ca(2+) concentrations in the physioloical range. The mechanism involved is due to a rise in the affinity of the Ca(2+) transport system, induced by polyamines, most probably exhibiting allosteric behaviour. The regulatory site of this mechanism is the so-called S(1) binding site of polyamines, which operates in physiological conditions and is located in the energy well between the two peaks present in the energy profile of mitochondrial spermine transport. Spermine is bidirectionally transported across teh inner membrane by cycling, in which influx and efflux are driven by electrical and pH gradients, respectively. Most probably, polyamine affects the Ca(2+) transport system when it acts from the outside-that is, in the direction of its uniporter channel, in order to reach the S(1) site. Important physiological functions are related to activation of Ca(2+) transport systems by polyamines and their interactions with the S(1) site. These functions include a rise in the metabolic rate for energy supply and modulation of mitochondrial permeability transition induction, with consequent effects on the triggering of the apoptotic pathway.     

Mitochondria and Ca(2+)in cell physiology and pathophysiology

There is now a consensus that mitochondria take up and accumulate Ca(2+)during physiological [Ca(2+)](c)signalling. This contribution will consider some of the functional consequences of mitochondrial Ca(2+)uptake for cell physiology and pathophysiology. The ability to remove Ca(2+)from local cytosol enables mitochondria to regulate the [Ca(2+)] in microdomains close to IP3-sensitive Ca(2+)-release channels. The [Ca(2+)] sensitivity of these channels means that, by regulating local [Ca(2+)](c), mitochondrial Ca(2+)uptake modulates the rate and extent of propagation of [Ca(2+)](c)waves in a variety of cell types. The coincidence of mitochondrial Ca(2+)uptake with oxidative stress may open the mitochondrial permeability transition pore (mPTP). This is a catastrophic event for the cell that will initiate pathways to cell death either by necrotic or apoptotic pathways. A model is presented in which illumination of an intramitochondrial fluorophore is used to generate oxygen radical species within mitochondria. This causes mitochondrial Ca(2+)loading from SR and triggers mPTP opening. In cardiomyocytes, mPTP opening leads to ATP consumption by the mitochondrial ATPase and so results in ATP depletion, rigor and necrotic cell death. In central mammalian neurons exposed to glutamate, a cellular Ca(2+)overload coincident with NO production also causes loss of mitochondrial potential and cell death, but mPTP involvement has proven more difficult to demonstrate unequivocally.     

L-Type Ca(2+) channel charge movement and intracellular Ca(2+) in skeletal muscle fibers from aging mice

In this work we tested the hypothesis that skeletal muscle fibers from aging mice exhibit a significant decline in myoplasmic Ca(2+) concentration resulting from a reduction in L-type Ca(2+) channel (dihydropyridine receptor, DHPR) charge movement. Skeletal muscle fibers from the flexor digitorum brevis (FDB) muscle were obtained from 5-7-, 14-18-, or 21-24-month-old FVB mice and voltage-clamped in the whole-cell configuration of the patch-clamp technique according to described procedures (Wang, Z.-M., M. L. Messi, and O. Delbono. 1999. Biophys. J. 77:2709-2716). Total charge movement or the DHPR charge movement was measured simultaneously with intracellular Ca(2+) concentration. The maximum charge movement (Q(max)) recorded (mean +/- SEM, in nC microF(-1)) was 53 +/- 3.2 (n = 47), 51 +/- 3.2 (n = 35) (non-significant, ns), and 33 +/- 1.9 (n = 32) (p < 0.01), for the three age groups, respectively. Q(max) corresponding to the DHPR was 43 +/- 3.3, 38 +/- 4.1 (ns), and 25 +/- 3.4 (p < 0.01) for the three age groups, respectively. The peak intracellular [Ca(2+)] recorded at 40 mV (in microM) was 15.7 +/- 0. 12, 16.7 +/- 0.18 (ns), and 8.2 +/- 0.07 (p < 0.01) for the three age groups, respectively. No significant changes in the voltage distribution or steepness of the Q-V or [Ca(2+)]-V relationship were found. These data support the concept that the reduction in the peak intracellular [Ca(2+)] results from a larger number of ryanodine receptors uncoupled to DHPRs in skeletal muscle fibers from aging mammals.     

Sarcoplasmic reticulum Ca(2+) release and muscle fatigue.

Efforts to examine the relevant mechanisms involved in skeletal muscle fatigue are focusing on Ca(2+) handling within the active muscle cell. It has been demonstrated time and again that reductions in sarcoplasmic reticulum (SR) Ca(2+) release resulting from increased or intense muscle contraction will compromise tension development. This review seeks to accomplish two related goals: 1) to provide an up-to-date molecular understanding of the Ca(2+)-release process, with considerable attention devoted to the SR Ca(2+) channel, including its associated proteins and their regulation by endogenous compounds; and 2) to examine several putative mechanisms by which cellular alterations resulting from intense and/or prolonged contractile activity will modify SR Ca(2+) release. The mechanisms that are likely candidates to explain the reductions in SR Ca(2+) channel function following contractile activity include elevated Ca(2+) concentrations, alterations in metabolic homeostasis within the "microcompartmentalized" triadic space, and modification by reactive oxygen species.     

Mitochondria fine-tune the slow Ca(2+) transients induced by electrical stimulation of skeletal myotubes

Mitochondria sense cytoplasmic Ca(2+) signals in many cell types. In mammalian skeletal myotubes, depolarizing stimuli induce two independent cytoplasmic Ca(2+) signals: a fast signal associated with contraction and a slow signal that propagates to the nucleus and regulates gene expression. How mitochondria sense and possibly affect these cytoplasmic Ca(2+) signals has not been reported. We investigated here (a) the emergence of mitochondrial Ca(2+) signals in response to electrical stimulation of myotubes, (b) the contribution of mitochondrial Ca(2+) transients to ATP generation and (c) the influence of mitochondria as modulators of cytoplasmic and nuclear Ca(2+) signals. Rhod2 and Fluo3 fluorescence determinations revealed composite Ca(2+) signals associated to the mitochondrial compartment in electrically stimulated (400 pulses, 45 Hz) skeletal myotubes. Similar Ca(2+) signals were detected when using a mitochondria-targeted pericam. The fast mitochondrial Ca(2+) rise induced by stimulation was inhibited by pre-incubation with ryanodine, whereas the phospholipase C inhibitor U73122 blocked the slow mitochondrial Ca(2+) signal, showing that mitochondria sense the two cytoplasmic Ca(2+) signal components. The fast but not the slow Ca(2+) transient enhanced mitochondrial ATP production. Inhibition of the mitochondrial Ca(2+) uniporter prevented the emergence of mitochondrial Ca(2+) transients and significantly increased the magnitude of slow cytoplasmic Ca(2+) signals after stimulation. Precluding mitochondrial Ca(2+) extrusion with the Na(+)/Ca(2+) exchanger inhibitor CGP37157 decreased mitochondrial potential, increased the magnitude of the slow cytoplasmic Ca(2+) signal and decreased the rate of Ca(2+) signal propagation from one nucleus to the next. Over expression of the mitochondrial fission protein Drp-1 decreased mitochondrial size and the slow Ca(2+) transient in mitochondria, but enhanced cytoplasmic and nuclear slow transients. The present results indicate that mitochondria play a central role in the regulation of Ca(2+) signals involved in gene expression in myotubes.     

Simulation of Ca2+ movements within the sarcomere of fast-twitch mouse fibers stimulated by action potentials

Ca(2+) release from the sarcoplasmic reticulum (SR) of skeletal muscle takes place at the triadic junctions; following release, Ca(2+) spreads within the sarcomere by diffusion. Here, we report multicompartment simulations of changes in sarcomeric Ca(2+) evoked by action potentials (APs) in fast-twitch fibers of adult mice. The simulations include Ca(2+) complexation reactions with ATP, troponin, parvalbumin, and the SR Ca(2+) pump, as well as Ca(2+) transport by the pump. Results are compared with spatially averaged Ca(2+) transients measured in mouse fibers with furaptra, a low-affinity, rapidly responding Ca(2+) indicator. The furaptra Deltaf(CaD) signal (change in the fraction of the indicator in the Ca(2+)-bound form) evoked by one AP is well simulated under the assumption that SR Ca(2+) release has a peak of 200-225 microM/ms and a FDHM of approximately 1.6 ms (16 degrees C). Deltaf(CaD) elicited by a five-shock, 67-Hz train of APs is well simulated under the assumption that in response to APs 2-5, Ca(2+) release decreases progressively from 0.25 to 0.15 times that elicited by the first AP, a reduction likely due to Ca(2+) inactivation of Ca(2+) release. Recovery from inactivation was studied with a two-AP protocol; the amplitude of the second release recovered to >0.9 times that of the first with a rate constant of 7 s(-1). An obvious feature of Deltaf(CaD) during a five-shock train is a progressive decline in the rate of decay from the individual peaks of Deltaf(CaD). According to the simulations, this decline is due to a reduction in available Ca(2+) binding sites on troponin and parvalbumin. The effects of sarcomere length, the location of the triadic junctions, resting [Ca(2+)], the parvalbumin concentration, and possible uptake of Ca(2+) by mitochondria were also investigated. Overall, the simulations indicate that this reaction-diffusion model, which was originally developed for Ca(2+) sparks in frog fibers, works well when adapted to mouse fast-twitch fibers stimulated by APs.     

Mitochondrial modulation of intracellular Ca(2+) signaling

IP3-mediated Ca(2+) release plays a fundamental role in many cell signaling processes and has been the subject of numerous modeling studies. Only recently has the important role that mitochondria play in the dynamics of intracellular Ca(2+) signaling begun to be considered in experimental work and in computational models. Mitochondria sequester large amounts of Ca(2+) and thus have a modulatory effect on intracellular Ca(2+) signaling, and mitochondrial uptake of Ca(2+), in turn, has a regulatory effect on mitochondrial function. Here we integrate a well-established model of IP3-mediated Ca(2+) signaling with a detailed model of mitochondrial Ca(2+) handling and metabolic function. The incorporation of mitochondria results in oscillations in a bistable formulation of the IP3 model, and increasing metabolic substrate decreases the frequency of these oscillations consistent with the literature. Ca(2+) spikes from the cytosol are communicated into mitochondria and are shown to induce realistic metabolic changes. The model has been formulated using a modular approach that is easy to modify and should serve as a useful basis for the investigation of questions regarding the interaction of these two systems.     

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.