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.     

Role of mitochondrial Ca2+ in the regulation of cellular energetics

Calcium is an important signaling molecule involved in the regulation of many cellular functions. The large free energy in the Ca(2+) ion membrane gradients makes Ca(2+) signaling inherently sensitive to the available cellular free energy, primarily in the form of ATP. In addition, Ca(2+) regulates many cellular ATP-consuming reactions such as muscle contraction, exocytosis, biosynthesis, and neuronal signaling. Thus, Ca(2+) becomes a logical candidate as a signaling molecule for modulating ATP hydrolysis and synthesis during changes in numerous forms of cellular work. Mitochondria are the primary source of aerobic energy production in mammalian cells and also maintain a large Ca(2+) gradient across their inner membrane, providing a signaling potential for this molecule. The demonstrated link between cytosolic and mitochondrial Ca(2+) concentrations, identification of transport mechanisms, and the proximity of mitochondria to Ca(2+) release sites further supports the notion that Ca(2+) can be an important signaling molecule in the energy metabolism interplay of the cytosol with the mitochondria. Here we review sites within the mitochondria where Ca(2+) plays a role in the regulation of ATP generation and potentially contributes to the orchestration of cellular metabolic homeostasis. Early work on isolated enzymes pointed to several matrix dehydrogenases that are stimulated by Ca(2+), which were confirmed in the intact mitochondrion as well as cellular and in vivo systems. However, studies in these intact systems suggested a more expansive influence of Ca(2+) on mitochondrial energy conversion. Numerous noninvasive approaches monitoring NADH, mitochondrial membrane potential, oxygen consumption, and workloads suggest significant effects of Ca(2+) on other elements of NADH generation as well as downstream elements of oxidative phosphorylation, including the F(1)F(O)-ATPase and the cytochrome chain. These other potential elements of Ca(2+) modification of mitochondrial energy conversion will be the focus of this review. Though most specific molecular mechanisms have yet to be elucidated, it is clear that Ca(2+) provides a balanced activation of mitochondrial energy metabolism that exceeds the alteration of dehydrogenases alone.     

The mechanism of lead-induced mitochondrial Ca2+ efflux

Addition of Pb²⁺ to rat kidney mitochondria is followed by induction of several reactions: inhibition of Ca²⁺ uptake, collapse of the transmembrane potential, oxidation of pyridine nucleotides, and a fast release of accumulated Ca²⁺. When the incubation media are supplemented with ruthenium red, the effect of Pb²⁺ on NAD(P)H oxidation, membrane , and Ca²⁺ release are not prevented if malate-glutamate are the oxidizing substrates; however, the latter two lead-induced reactions are prevented by ruthenium red if succinate is the electron donor. It is proposed that in mitochondria oxidizing NAD-dependent substrates, Pb²⁺ induces Ca²⁺ release by promoting NAD(P)H oxidation and a parallel drop in due to its binding to thiol groups, located in the cytosol side of the inner membrane. In addition, it is proposed that with succinate as substrate, the Ca²⁺-releasing effect of lead is due to the collapse of the transmembrane potential as a consequence of the uptake of Pb²⁺ through the calcium uniporter, since such effect is ruthenium red sensitive.     

Store-operated Ca2+ entry depends on mitochondrial Ca2+ uptake

Store-operated Ca(2+) channels, which are activated by the emptying of intracellular Ca(2+) stores, provide one major route for Ca(2+) influx. Under physiological conditions of weak intracellular Ca(2+) buffering, the ubiquitous Ca(2+) releasing messenger InsP(3) usually fails to activate any store-operated Ca(2+) entry unless mitochondria are maintained in an energized state. Mitochondria rapidly take up Ca(2+) that has been released by InsP(3), enabling stores to empty sufficiently for store-operated channels to activate. Here, we report a novel role for mitochondria in regulating store-operated channels under physiological conditions. Mitochondrial depolarization suppresses store-operated Ca(2+) influx independently of how stores are depleted. This role for mitochondria is unrelated to their actions on promoting InsP(3)-sensitive store depletion, can be distinguished from Ca(2+)-dependent inactivation of the store-operated channels and does not involve changes in intracellular ATP, oxidants, cytosolic acidification, nitric oxide or the permeability transition pore, but is suppressed when mitochondrial Ca(2+) uptake is impaired. Our results suggest that mitochondria may have a more fundamental role in regulating store-operated influx and raise the possibility of bidirectional Ca(2+)-dependent crosstalk between mitochondria and store-operated Ca(2+) channels.     

Silymarin-induced mitochondrial Ca2+ release

The effect of silymarin on different functions of mitochondria isolated from rat kidneys was studied. Addition of silymarin to mitochondria oxidizing succinate, induced stimulation of the respiratory State 4; while in mitochondria oxidizing NAD-dependent substrates, the drug produced inhibition of the oxygen consumption. It is also shown that silymarin induces mitochondrial swelling, a drop in the transmembrane potential, as well as Ca2+ release. It is proposed that due to its hydrophobic character, silymarin produces an alteration in the lipidic milieu of the inner membrane which is conductive to an inhibition of the electron transport in the NAD-CoQ span of the respiratory chain, as well as to the loss of the energy dependent accumulated Ca2+.     

Dynamic buffering of mitochondrial Ca2+ during Ca2+ uptake and Na+-induced Ca2+ release

In cardiac mitochondria, matrix free Ca²⁺ ([Ca²⁺]_m) is primarily regulated by Ca²⁺ uptake and release via the Ca²⁺ uniporter (CU) and Na⁺/Ca²⁺ exchanger (NCE) as well as by Ca²⁺ buffering. Although experimental and computational studies on the CU and NCE dynamics exist, it is not well understood how matrix Ca²⁺ buffering affects these dynamics under various Ca²⁺ uptake and release conditions, and whether this influences the stoichiometry of the NCE. To elucidate the role of matrix Ca²⁺ buffering on the uptake and release of Ca²⁺, we monitored Ca²⁺ dynamics in isolated mitochondria by measuring both the extra-matrix free [Ca²⁺] ([Ca²⁺]_e) and [Ca²⁺]_m. A detailed protocol was developed and freshly isolated mitochondria from guinea pig hearts were exposed to five different [CaCl₂] followed by ruthenium red and six different [NaCl]. By using the fluorescent probe indo-1, [Ca²⁺]_e and [Ca²⁺]_m were spectrofluorometrically quantified, and the stoichiometry of the NCE was determined. In addition, we measured NADH, membrane potential, matrix volume and matrix pH to monitor Ca²⁺-induced changes in mitochondrial bioenergetics. Our [Ca²⁺]_e and [Ca²⁺]_m measurements demonstrate that Ca²⁺ uptake and release do not show reciprocal Ca²⁺ dynamics in the extra-matrix and matrix compartments. This salient finding is likely caused by a dynamic Ca²⁺ buffering system in the matrix compartment. The Na⁺- induced Ca²⁺ release demonstrates an electrogenic exchange via the NCE by excluding an electroneutral exchange. Mitochondrial bioenergetics were only transiently affected by Ca²⁺ uptake in the presence of large amounts of CaCl₂, but not by Na⁺- induced Ca²⁺ release.     

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.     

Mitochondrial membrane potential modulates regulation of mitochondrial Ca2+ in rat ventricular myocytes

Although recent studies focused on the contribution of mitochondrial Ca2+ to the mechanisms of ischemia-reperfusion injury, the regulation of mitochondrial Ca2+ under pathophysiological conditions remains largely unclear. By using saponin-permeabilized rat myocytes, we measured mitochondrial membrane potential (DeltaPsi(m)) and mitochondrial Ca2+ concentration ([Ca2+](m)) at the physiological range of cytosolic Ca2+ concentration ([Ca2+](c); 300 nM) and investigated the regulation of [Ca2+](m) during both normal and dissipated DeltaPsi(m). When DeltaPsi(m) was partially depolarized by carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP, 0.01-0.1 microM), there were dose-dependent decreases in [Ca2+](m). When complete DeltaPsi(m) dissipation was achieved by FCCP (0.3-1 microM), [Ca2+](m) remained at one-half of the control level despite no Ca2+ influx via the Ca2+ uniporter. The DeltaPsi(m) dissipation by FCCP accelerated calcein leakage from mitochondria in a cyclosporin A (CsA)-sensitive manner, which indicates that DeltaPsi(m) dissipation opened the mitochondrial permeability transition pore (mPTP). After FCCP addition, inhibition of the mPTP by CsA caused further [Ca2+](m) reduction; however, inhibition of mitochondrial Na+/Ca2+ exchange (mitoNCX) by a Na+-free solution abolished this [Ca2+](m) reduction. Cytosolic Na(+) concentrations that yielded one-half maximal activity levels for mitoNCX were 3.6 mM at normal DeltaPsi(m) and 7.6 mM at DeltaPsi(m) dissipation. We conclude that 1) the mitochondrial Ca2+ uniporter accumulates Ca2+ in a manner that is dependent on DeltaPsi(m) at the physiological range of [Ca2+](c); 2) DeltaPsi(m) dissipation opens the mPTP and results in Ca2+ influx to mitochondria; and 3) although mitoNCX activity is impaired, mitoNCX extrudes Ca2+ from the matrix even after DeltaPsi(m) dissipation.     

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.     

Unified mechanisms of Ca2+ regulation across the Ca2+ channel family

L-type (CaV1.2) and P/Q-type (CaV2.1) calcium channels possess lobe-specific CaM regulation, where Ca2+ binding to one or the other lobe of CaM triggers regulation, even with inverted polarity of modulation between channels. Other major members of the CaV1-2 channel family, R-type (CaV2.3) and N-type (CaV2.2), have appeared to lack such CaM regulation. We report here that R- and N-type channels undergo Ca(2+)-dependent inactivation, which is mediated by the CaM N-terminal lobe and present only with mild Ca2+ buffering (0.5 mM EGTA) characteristic of many neurons. These features, together with the CaM regulatory profiles of L- and P/Q-type channels, are consistent with a simplifying principle for CaM signal detection in CaV1-2 channels-independent of channel context, the N- and C-terminal lobes of CaM appear invariably specialized for decoding local versus global Ca2+ activity, respectively.     

Redox regulation of ER and mitochondrial Ca2+ signaling in cell survival and death

Physiological signaling by reactive oxygen species (ROS) and their pathophysiological role in cell death are well recognized. This review focuses on two ROS targets that are key to local Ca²⁺ signaling at the ER/mitochondrial interface – notably, inositol trisphosphate receptors (IP₃Rs) and the mitochondrial calcium uniporter (MCU). Both transport systems are central to molecular mechanisms in cell survival and death. Methods for the measurement of the redox state of these proteins and for the detection of ROS nanodomains are described. Recent results on the redox regulation of these proteins are reviewed.     

Physiological role of mitochondrial Ca2+ transport

A model has been proposed in which mitochondrial Ca²⁺ ion transport serves to regulate mitochondrial matrix free Ca²⁺ ([Ca²⁺]_m), with the advantage to the animal that this allows the regulation of pyruvate dehydrogenase and the tricarboxylate cycle in response to energy demand. This article examines recent evidence for dehydrogenase activation and for increases in [Ca²⁺]_m in response to increased tissue energy demands, especially in cardiac myocytes and in heart. It critiques recent results on beat-to-beat variation in [Ca²⁺]_m in cardiac muscle and also briefly surveys the impact of mitochondrial Ca^2– transport on transient changes in cytosolic free Ca²⁺ in excitable tissues. Finally, it proposes that a failure to elevate [Ca²⁺]_m sufficiently in response to work load may underlie some cardiomyopathies of metabolic origin.     

The role of mitochondrial Ca2+ transport and matrix Ca2+ in signal transduction in mammalian tissues

The pyruvate, NAD(+)-isocitrate and 2-oxoglutarate dehydrogenases are key regulatory enzymes in intramitochondrial oxidative metabolism in mammalian tissues, and can all be activated by increases in Ca2+ in the micromolar range. There is now mounting evidence that hormones and other stimuli which act by increasing cytosolic Ca2+ also, as a result, cause increases in mitochondrial matrix Ca2+ and hence activation of these enzymes, suggesting that the primary physiological function of mitochondrial Ca2(+)-transport is to be involved in this relay mechanism. This may also explain how in such circumstances rates of ATP production may be increased to meet the greater demand, but without any decreases in ATP/ADP occurring.     

Ca2+ regulation in the Na+/Ca2+ exchanger involves two markedly different Ca2+ sensors

The plasma membrane Na+/Ca2+ exchanger (NCX) is almost certainly the major Ca2+ extrusion mechanism in cardiac myocytes. Binding of Na+ and Ca2+ ions to its large cytosolic loop regulates ion transport of the exchanger. We determined the solution structures of two Ca2+ binding domains (CBD1 and CBD2) that, together with an alpha-catenin-like domain (CLD), form the regulatory exchanger loop. CBD1 and CBD2 are very similar in the Ca2+ bound state and describe the Calx-beta motif. Strikingly, in the absence of Ca2+, the upper half of CBD1 unfolds while CBD2 maintains its structural integrity. Together with a 7-fold higher affinity for Ca2+, this suggests that CBD1 is the primary Ca2+ sensor. Specific point mutations in either domain largely allow the interchange of their functionality and uncover the mechanism underlying Ca2+ sensing in NCX.     

Sustained Ca2+ transfer across mitochondria is Essential for mitochondrial Ca2+ buffering,sore-operated Ca2+ entry,and Ca2+ store refilling

Mitochondria have been found to sequester and release Ca2+ during cell stimulation with inositol 1,4,5-triphosphate-generating agonists, thereby generating subplasmalemmal microdomains of low Ca2+ that sustain activity of capacitative Ca2+ entry (CCE). Procedures that prevent mitochondrial Ca2+ uptake inhibit local Ca2+ buffering and CCE, but it is not clear whether Ca2+ has to transit through or remains trapped in the mitochondria. Thus, we analyzed the contribution of mitochondrial Ca2+ efflux on the ability of mitochondria to buffer subplasmalemmal Ca2+, to maintain CCE, and to facilitate endoplasmic reticulum (ER) refilling in endothelial cells. Upon the addition of histamine, the initial mitochondrial Ca2+ transient, monitored with ratio-metric-pericam-mitochondria, was largely independent of extracellular Ca2+. However, subsequent removal of extracellular Ca2+ produced a reversible decrease in [Ca2+]mito, indicating that Ca2+ was continuously taken up and released by mitochondria, although [Ca2+]mito had returned to basal levels. Accordingly, inhibition of the mitochondrial Na+/Ca2+ exchanger with CGP 37157 increased [Ca2+]mito and abolished the ability of mitochondria to buffer subplasmalemmal Ca2+, resulting in an increased activity of BKCa channels and a decrease in CCE. Hence, CGP 37157 also reversibly inhibited ER refilling during cell stimulation. These effects of CGP 37157 were mimicked if mitochondrial Ca2+ uptake was prevented with oligomycin/antimycin A. Thus, during cell stimulation a continuous Ca2+ flux through mitochondria underlies the ability of mitochondria to generate subplasmalemmal microdomains of low Ca2+, to facilitate CCE, and to relay Ca2+ from the plasma membrane to the ER.     

Intraluminal Ca2+ dependence of Ca2+ and ryanodine-mediated regulation of skeletal muscle sarcoplasmic reticulum Ca2+ release.

The action of ryanodine upon sarcoplasmic reticulum (SR) Ca2+ handling is controversial with evidence for both activation and inhibition of SR Ca2+ release. In this study, the role of the intraluminal SR Ca2+ load was probed as a potential regulator of ryanodine-mediated effects upon SR Ca2+ release. Through dual-wavelength spectroscopy of Ca2+:antipyrylazo III difference absorbance, the intraluminal Ca2+ dependence of ryanodine and Ca(2+)-induced Ca2+ release (CICR) from skeletal SR vesicles was examined. Ryanodine addition after initiation of Ca2+ uptake (a) increased the intraluminal Ca2+ sensitivity of CICR and (b) stimulated spontaneous Ca2+ release with a delayed onset. These ryanodine effects were inversely proportional to the intraluminal Ca2+ load. Ryanodine also inhibited subsequent CICR after reaccumulation of Ca2+ released from the initial CICR. These results provide evidence that ryanodine inhibits transitions between low and high affinity Ca2+ binding states of an intraluminal Ca2+ compartment, possibly calsequestrin. Conformational transitions of calsequestrin may be reciprocally coupled to transitions between open and closed states of the Ca2+ release channel.