Mitochondria extrude protons across their inner membrane to generate the mitochondrial membrane potential (ΔΨm) and pH gradient (ΔpHm) that both power ATP synthesis. Mitochondrial uptake and efflux of many ions and metabolites are driven exclusively by ΔpHm, whose in situ regulation is poorly characterized. Here, we report the first dynamic measurements of ΔpHm in living cells, using a mitochondrially targeted, pH-sensitive YFP (SypHer) combined with a cytosolic pH indicator (5-(and 6)-carboxy-SNARF-1). The resting matrix pH (∼7.6) and ΔpHm (∼0.45) of HeLa cells at 37 °C were lower than previously reported. Unexpectedly, mitochondrial pH and ΔpHm decreased during cytosolic Ca2+ elevations. The drop in matrix pH was due to cytosolic acid generated by plasma membrane Ca2+-ATPases and transmitted to mitochondria by Pi/H+ symport and K+/H+ exchange, whereas the decrease in ΔpHm reflected the low H+-buffering power of mitochondria (∼5 mm, pH 7.8) compared with the cytosol (∼20 mm, pH 7.4). Upon agonist washout and restoration of cytosolic Ca2+ and pH, mitochondria alkalinized and ΔpHm increased. In permeabilized cells, a decrease in bath pH from 7.4 to 7.2 rapidly decreased mitochondrial pH, whereas the addition of 10 μm Ca2+ caused a delayed and smaller alkalinization. These findings indicate that the mitochondrial matrix pH and ΔpHm are regulated by opposing Ca2+-dependent processes of stimulated mitochondrial respiration and cytosolic acidification.

The ability of mitochondria to capture Ca2+ ions has important functional implications for cells, because mitochondria shape cellular Ca2+ signals by acting as a Ca2+ buffer and respond to Ca2+ elevations either by increasing the cell energy supply or by triggering the cell death program of apoptosis. A mitochondrial Ca2+ channel known as the uniporter drives the rapid and massive entry of Ca2+ ions into mitochondria. The uniporter operates at high, micromolar cytosolic Ca2+ concentrations that are only reached transiently in cells, near Ca2+ release channels. Mitochondria can also take up Ca2+ at low, nanomolar concentrations, but this high affinity mode of Ca2+ uptake is not well characterized. Recently, leucine-zipper-EF hand-containing transmembrane region (Letm1) was proposed to be an electrogenic 1:1 mitochondrial Ca2+/H+ antiporter that drives the uptake of Ca2+ into mitochondria at nanomolar cytosolic Ca2+ concentrations. In this article, we will review the properties of the Ca2+ import systems of mitochondria and discuss how Ca2+ uptake via an electrogenic 1:1 Ca2+/H+ antiport challenges our current thinking of the mitochondrial Ca2+ uptake mechanism.

The chemical nature and functional significance of mitochondrial flashes associated with fluctuations in mitochondrial membrane potential is unclear. Using a ratiometric pH probe insensitive to superoxide, we show that flashes reflect matrix alkalinization transients of ∼0.4 pH units that persist in cells permeabilized in ion-free solutions and can be evoked by imposed mitochondrial depolarization. Ablation of the pro-fusion protein Optic atrophy 1 specifically abrogated pH flashes and reduced the propagation of matrix photoactivated GFP (paGFP). Ablation or invalidation of the pro-fission Dynamin-related protein 1 greatly enhanced flash propagation between contiguous mitochondria but marginally increased paGFP matrix diffusion, indicating that flashes propagate without matrix content exchange. The pH flashes were associated with synchronous depolarization and hyperpolarization events that promoted the membrane potential equilibration of juxtaposed mitochondria. We propose that flashes are energy conservation events triggered by the opening of a fusion pore between two contiguous mitochondria of different membrane potentials, propagating without matrix fusion to equilibrate the energetic state of connected mitochondria.

Medium-chain triglycerides have been used as part of a ketogenic diet effective in reducing epileptic episodes. The health benefits of the derived medium-chain fatty acids (MCFAs) are thought to result from the stimulation of liver ketogenesis providing fuel for the brain. We tested whether MCFAs have direct effects on energy metabolism in induced pluripotent stem cell-derived human astrocytes and neurons. Using single-cell imaging, we observed an acute pronounced reduction of the mitochondrial electrical potential and a concomitant drop of the NAD(P)H signal in astrocytes, but not in neurons. Despite the observed effects on mitochondrial function, MCFAs did not lower intracellular ATP levels or activate the energy sensor AMP-activated protein kinase. ATP concentrations in astrocytes were unaltered, even when blocking the respiratory chain, suggesting compensation through accelerated glycolysis. The MCFA decanoic acid (300 μM) promoted glycolysis and augmented lactate formation by 49.6%. The shorter fatty acid octanoic acid (300 μM) did not affect glycolysis but increased the rates of astrocyte ketogenesis 2.17-fold compared with that of control cells. MCFAs may have brain health benefits through the modulation of astrocyte metabolism leading to activation of shuttle systems that provide fuel to neighboring neurons in the form of lactate and ketone bodies.-Thevenet, J., De Marchi, U., Santo Domingo, J., Christinat, N., Bultot, L., Lefebvre, G., Sakamoto, K., Descombes, P., Masoodi, M., Wiederkehr, A. Medium-chain fatty acids inhibit mitochondrial metabolism in astrocytes promoting astrocyte-neuron lactate and ketone body shuttle systems.

During cell activation, mitochondria play an important role in Ca2+ homoeostasis due to the presence of a fast and specific Ca2+ channel in its inner membrane, the mitochondrial Ca2+ uniporter. This channel allows mitochondria to buffer local cytosolic [Ca2+] changes and controls the intramitochondrial Ca2+ levels, thus modulating a variety of phenomena from respiratory rate to apoptosis. We have described recently that SB202190, an inhibitor of p38 MAPK (mitogen-activated protein kinase), strongly activated the uniporter. We show in the present study that a series of natural plant flavonoids, widely distributed in foods, produced also a strong stimulation of the mitochondrial Ca2+ uniporter. This effect was of the same magnitude as that induced by SB202190 (an approx. 20-fold increase in the mitochondrial Ca2+ uptake rate), developed without measurable delay and was rapidly reversible. In intact cells, the mitochondrial Ca2+ peak induced by histamine was also largely increased by the flavonoids. Stimulation of the uniporter by either flavonoids or SB202190 did not require ATP, suggesting a direct effect on the uniporter or an associated protein which is not mediated by protein phosphorylation. The most active compound, kaempferol, increased the rate of mitochondrial Ca2+ uptake by 85+/-15% (mean+/-S.E.M., n=4) and the histamine-induced mitochondrial Ca2+ peak by 139+/-19% (mean+/-S.E.M., n=5) at a concentration of 1 microM. Given that flavonoids can reach this concentration range in plasma after ingestion of flavonoid-rich food, these compounds could be modulating the uniporter under physiological conditions.

The generation of a proton gradient across the inner mitochondrial membrane (IMM) is an essential energy conservation event that couples the oxidation of carbohydrates and fat to the synthesis of ATP. Studies in isolated mitochondria have established that the chemical gradient for protons (pH m ) and the mitochondrial membrane potential ( m ) contribute independently to the proton-motive force (p) that drives the synthesis of ATP. Because  m contributes most of the p and can be easily measured in intact cells with fluorescent dyes, most studies ignore the contribution of pH m and only record changes in  m to track the metabolic state of mitochondria. pH m , however, drives the fluxes of metabolic substrates required for mitochondrial respiration and the activity of electroneutral ion exchangers that maintain mitochondria osmolarity and volume, and recent studies indicate that the mitochondrial pH (pH mito ) plays an important and underappreciated role in physiological and pathological situations such as apoptosis, neurotransmission, and insulin secretion. In this Perspective, we discuss the putative roles of the pH mito and review the different techniques used to measure pH mito and pH m in isolated mitochondria and in intact cells, focusing on our recent results obtained with genetically encoded pH-sensitive indicators. These measurements have revealed that the pH mito is in dynamic equilibrium with the cytosolic pH and that spontaneous pH mito elevations coinciding with  m drops occur in single mitochondria. Unlike the "superoxide flashes" reported with a pH-sensitive circularly permuted YFP (cpYFP), these "pH flashes" preserve the p during spontaneous fluctuations in  m ; therefore, we propose that the flashes are energy conservation events that reflect the intrinsic properties of the mitochondrial proton circuit.

Background: Whether mitochondrial Ca 2ϩ extrusion is mediated by NCLX (mitochondrial sodium/calcium exchanger) or LETM1 (leucine zipper-EF-hand-containing transmembrane protein 1) and controls matrix redox state is unknown. Results: NCLX, but not LETM1, increases Ca 2ϩ extrusion, limits NAD(P)H production, and promotes matrix oxidation. Conclusion: NCLX controls the duration of matrix Ca 2ϩ elevations and their impact on redox signaling. Significance: NCLX is a potential target for the treatment of redox-dependent diseases.

Background and purpose: The thiourea derivative KB-R7943, originally developed as inhibitor of the plasma membrane Na þ /Ca 2 þ exchanger, has been shown to protect against myocardial ischemia-reperfusion injury. We have studied here its effects on mitochondrial Ca 2 þ fluxes. Experimental approach. [Ca 2 þ ] in cytosol, mitochondria and endoplasmic reticulum (ER), and mitochondrial membrane potential were monitored using both luminescent (targeted aequorins) and fluorescent (fura-2, tetramethylrhodamine ethyl ester) probes in HeLa cells. Key results: KB-R7943 was also a potent inhibitor of the mitochondrial Ca 2 þ uniporter (MCU). In permeabilized HeLa cells, KB-R7943 inhibited mitochondrial Ca 2 þ uptake with a Ki of 5.571.3 mM (mean7S.D.). In intact cells, 10mM KB-R7943 reduced by 80% the mitochondrial [Ca 2 þ ] peak induced by histamine. KB-R7943 did not modify the mitochondrial membrane potential and had no effect on the mitochondrial Na þ /Ca 2 þ exchanger. KB-R7943 inhibited histamine-induced ER-Ca 2 þ release in intact cells, but not in cells loaded with a Ca 2 þ -chelator to damp cytosolic [Ca 2 þ ] changes. Therefore, inhibition of ER-Ca 2 þ -release by KB-R7943 was probably due to the increased feedback Ca 2 þ -inhibition of inositol 1,4,5-trisphosphate receptors after MCU block. This mechanism also explains why KB-R7943 reversibly blocked histamine-induced cytosolic [Ca 2 þ ] oscillations in the same range of concentrations required to inhibit MCU. Conclusions and Implications: Inhibition of MCU by KB-R7943 may contribute to its cardioprotective activity by preventing mitochondrial Ca 2 þ -overload during ischemia-reperfusion. In addition, the effects of KB-R7943 on Ca 2 þ homeostasis provide new evidence for the role of mitochondria modulating Ca 2 þ -release and regenerative Ca 2 þ -oscillations. Search for permeable and selective MCU inhibitors may yield useful pharmacological tools in the future.