Regulation of mitochondrial ATP synthesis by calcium: Evidence for a long-term metabolic priming

Abstract

A TP is the primary utilizable source of high-energy phosphate bonds within the cell and acts as an allosteric effector of numerous cell processes. Most intracellular ATP is derived from cytosolic glycolysis and mitochondrial oxidative phosphorylation. The latter process couples the oxidation of reduced cofactors via the respiratory chain to ATP synthesis by mitochondrial ATP synthase. The supply of reduced cofactors (NADH, FADH 2 ) is ensured by mitochondrial oxidation of substrates derived from glucose, fatty acids, and amino acids via different metabolic pathways. Therefore, oxidative phosphorylation is a complex process regulated at different levels by the interactions of mitochondrial and cytosolic metabolism (1). In this crosstalk, mitochondrial Ca 2ϩ homeostasis, a process that has attracted a large interest in the past few years (2-11), appears to play a major role. Indeed, three dehydrogenases of the Krebs cycle (pyruvate, isocitrate, and ␣-ketoglutarate dehydrogenase) are modulated by [Ca 2ϩ ] in the micromolar range (12, 13). Recently, it has been shown that, despite the low affinity of the mitochondrial Ca 2ϩ uptake systems, large increases in matrix [Ca 2ϩ ] parallel the cytosolic Ca 2ϩ signals, thanks to the close contact between mitochondria and the intracellular Ca 2ϩ stores (14). The final outcome of this Ca 2ϩ transfer is expected to be the enhancement of mitochondrial ATP production to balance the increased ATP demand of a stimulated cell (15)(16)(17)(18).In this paper, we directly addressed these issues by investigating, in intact living cells, the effect of cytosolic and mitochondrial Ca 2ϩ signaling on intramitochondrial ATP concentration. For this purpose, we have utilized a targeted recombinant Ca 2ϩ probe (mitochondrial aequorin, mtAEQ) (2) and constructed a tool, a specifically targeted chimera of the ATP-sensitive photoprotein luciferase, with the aim of dynamically monitoring the ATP concentration in the mitochondrial matrix ([ATP] m ). The basis for this approach to the measurement of this key cellular parameter was the observation that the affinity of luciferase for ATP, which in vitro is in the micromolar range, is drastically lower in vivo, presumably because of the presence of competing proteins and anions (19). Indeed, in the cellular environment and in the presence of luciferin, luciferase light emission is a linear function of [ATP] in a concentration range between 10 Ϫ3 and 10 Ϫ2 M, i.e., in the physiological range (20,21). We thus constructed a chimeric cDNA, which allows the selective targeting of luciferase to the mitochondrial matrix. With this tool, we could not only directly demonstrate that agonist-dependent changes in mitochondrial Ca 2ϩ concentration correlate with an enhancement in mitochondrial ATP concentration, provided that oxidizable substrates are available, but also identify a phenomenon of long-term memory, allowing a prolonged metabolic ''priming'' that lasts longer than the mitochondrial [Ca 2ϩ ] increase. These observations may clarify how mitoch...

Role of the c subunit of the FOATP synthase in mitochondrial permeability transition

“…50 This choice stemmed from the fact that the activity of the PTPC is modulated by various intermediate metabolites, including NADH, ATP, ADP and inorganic phosphate. 9 As the c subunit is coded by three distinct genes (ATP5G1, ATP5G2 and ATP5G3) that generate polypeptides bearing different mitochondrial import sequences but forming the very same mature protein, 45 we had to envision a depletion strategy based on a mix of three commercial validated small-interfering RNAs (siRNAs) (Fig.…”

“…9 We are confident that this is not the case since: (1) The depletion of the F O c subunit in a nearto-purely glycolytic cell model, i.e., human cervical carcinoma HeLa cells, 50 failed to alter the levels of mitochondrial ATP synthesis, yet inhibited MPT, mitochondrial fragmentation and cell death as triggered by cytosolic Ca 2+ overload or oxidative stress; and (2) The downregulation of the F 1 α subunit (which-similar to the F O c subunit-is required for the catalytic activity of the F 1 F O ATP synthase) 62 completely failed to affect MPT and its functional consequences.…”

P2X7: a growth-promoting receptor—implications for cancer

“…There is a delicate relationship between Ca 2+ and mitochondria in that on the one hand a higher mitochondrial potential enhances the electrochemical gradient for Ca 2+ import into the mitochondrial matrix, and on the other, small increases in the mitochondrial Ca 2+ concentration cause hyperpolarization [25]. Mitochondrial Ca 2+ in P2X 7 -transfected cells turns out to be at least twice as high as in mock-transfected or wt cells, and also intramitochondrial Ca 2+ transients triggered by activation of plasma membrane receptors are several-fold higher than in control cells.…”

Bcl-2 and Ca2+ homeostasis in the endoplasmic reticulum

“…On the one hand, by stimulating Ca 2 þ -dependent enzymes [27][28][29] or metabolite carriers 30 it can stimulate aerobic metabolism 31 and enhance ATP production. 32 On the other hand, Ca 2 þ may synergize with apoptotic mediators and induce large-scale morphological changes of the organelle, possibly by favouring the opening of the large-conductance channel known as permeability transition pore (PTP), 33 and thus induce the release of mitochondrial proapoptotic factors. Not surprisingly, loading of Ca 2 þ in the matrix, and the opening of PTP, has been implicated in the pathogenesis of numerous human disorders, stemming from heart damage upon reperfusion to the pathogenesis of muscular dystrophies.…”