The homeostasis of the transmembrane potential of hydrogen ions in mitochondria is a prerequisite for the normal mitochondrial functioning. However, in different pathological conditions it is advisable to slightly reduce the membrane potential, while maintaining it at levels sufficient to produce ATP that will ensure the normal functioning of the cell. A number of chemical agents have been found to provide mild uncoupling; however, natural proteins residing in mitochondrial membrane can carry this mission, such as proteins from the UCP family, an adenine nucleotide translocator and a dicarboxylate carrier. In this study, we demonstrated that the butyl ester of rhodamine 19, C4R1, binds to the components of the mitochondrial ATP synthase complex due to electrostatic interaction and has a good uncoupling effect. The more hydrophobic derivative C12R1 binds poorly to mitochondria with less uncoupling activity. Mass spectrometry confirmed that C4R1 binds to the β-subunit of mitochondrial ATP synthase and based on molecular docking, a C4R1 binding model was constructed suggesting the binding site on the interface between the α- and β-subunits, close to the anionic amino acid residues of the β-subunit. The association of the uncoupling effect with binding suggests that the ATP synthase complex can provide induced uncoupling.

BACKGROUND: Cytoprotection afforded by mitochondrial ATP-sensitive K+-channel (mKATP-channel) opener diazoxide (DZ) largely depends on the activation of potassium cycle with eventual modulation of mitochondrial functions and ROS production. However, generally these effects were studied in the presence of Mg∙ATP known to block K+ transport. Thus, the purpose of our work was the estimation of DZ effects on K+ transport, K+ cycle and ROS production in rat liver mitochondria in the absence of Mg∙ATP.
RESULTS: Without Mg·ATP, full activation of native mKATP-channel, accompanied by the increase in ATP-insensitive K+ uptake, activation of K+-cycle and respiratory uncoupling, was reached at ≤0.5 μM of DZ,. Higher diazoxide concentrations augmented ATP-insensitive K+ uptake, but not mKATP-channel activity. mKATP-channel was blocked by Mg·ATP, reactivated by DZ, and repeatedly blocked by mKATP-channel blockers glibenclamide and 5-hydroxydecanoate, whereas ATP-insensitive potassium transport was blocked by Mg2+ and was not restored by DZ. High sensitivity of potassium transport to DZ in native mitochondria resulted in suppression of mitochondrial ROS production caused by the activation of K+-cycle on sub-micromolar scale. Based on the oxygen consumption study, the share of mKATP-channel in respiratory uncoupling by DZ was found.
CONCLUSIONS: The study of mKATP-channel activation by diazoxide in the absence of MgATP discloses novel, not described earlier, aspects of mKATP-channel interaction with this drug. High sensitivity of mKATP-channel to DZ results in the modulation of mitochondrial functions and ROS production by DZ on sub-micromolar concentration scale. Our experiments led us to the hypothesis that under the conditions marked by ATP deficiency affinity of mKATP-channel to DZ can increase, which might contribute to the high effectiveness of this drug in cardio- and neuroprotection.

Mitochondrial dysfunction plays a crucial role in the macroautophagy/autophagy cascade. In a recently published study Sun et al. described the induction of autophagy by the membranophilic triphenylphosphonium (TPP)-based cation 10-(6\'-ubiquinonyl) decyltriphenylphosphonium (MitoQ) in HepG2 cells (Sun C, et al. \"MitoQ regulates autophagy by inducing a pseudo-mitochondrial membrane potential [PMMP]\", Autophagy 2017, 13:730-738.). Sun et al. suggested that MitoQ adsorbed to the inner mitochondrial membrane with its cationic moiety remaining in the intermembrane space, adding a large number of positive charges and establishing a \"pseudo-mitochondrial membrane potential,\" which blocked the ATP synthase. Here we argue that the suggested mechanism for generation of the \"pseudo-mitochondrial membrane potential\" is physically implausible and contradicts earlier findings on the electrophoretic displacements of membranophilic cations within and through phospholipid membranes. We provide evidence that TPP-cations dissipated the mitochondrial membrane potential in HepG2 cells and that the induction of autophagy in carcinoma cells by TPP-cations correlated with the uncoupling of oxidative phosphorylation. The mild uncoupling of oxidative phosphorylation by various mitochondria-targeted penetrating cations may contribute to their reported therapeutic effects via inducing both autophagy and mitochondria-selective mitophagy.

The enzymes of oxidative phosphorylation is a striking example of the functional association of multiple enzyme complexes, working together to form ATP from cellular reducing equivalents. These complexes, such as cytochrome c oxidase or the ATP synthase, are typically investigated individually and therefore, their functional interplay is not well understood. Here, we present methodology that allows the co-reconstitution of purified terminal oxidases and ATP synthases in synthetic liposomes. The enzymes are functionally coupled via proton translocation where upon addition of reducing equivalents the oxidase creates and maintains a transmembrane electrochemical proton gradient that energizes the synthesis of ATP by the F1F0 ATP synthase. The method has been tested with the ATP synthases from Escherichia coli and spinach chloroplasts, and with the quinol and cytochrome c oxidases from E. coli and Rhodobacter sphaeroides, respectively. Unlike in experiments with the ATP synthase reconstituted alone, the setup allows in vitro ATP synthesis under steady state conditions, with rates up to 90 ATP×s(-1)×enzyme(-1). We have also used the novel system to study the phenomenon of \"mild uncoupling\" as observed in mitochondria upon addition of low concentrations of ionophores (e.g. FCCP, SF6847) and the recoupling effect of 6-ketocholestanol. While we could reproduce the described effects, our data with the in vitro system does not support the idea of a direct interaction between a mitochondrial protein and the uncoupling agents as proposed earlier.

Mitochondrial uncoupling reduces reactive oxygen species (ROS) production and appears to be important for cellular signaling/protection, making it a focus for the treatment of metabolic and age-related diseases. Whereas the physiological role of uncoupling protein 1 (UCP1) of brown adipose tissue is established for thermogenesis, the function of UCP1 in the reduction of ROS in cold-exposed animals is currently under debate. Here, we investigated the role of UCP1 in mitochondrial ROS handling in the Lesser hedgehog tenrec (Echinops telfairi), a unique protoendothermic Malagasy mammal with recently identified brown adipose tissue (BAT). We show that the reduction of ROS by UCP1 activity also occurs in BAT mitochondria of the tenrec, suggesting that the antioxidative role of UCP1 is an ancient mammalian trait. Our analysis shows that the quantity of UCP1 displays strong control over mitochondrial hydrogen peroxide release, whereas other factors, such as mild cold, nonshivering thermogenesis, oxidative capacity, and mitochondrial respiration, do not correlate. Furthermore, hydrogen peroxide release from recoupled BAT mitochondria was positively associated with mitochondrial membrane potential. These findings led to a model of UCP1 controlling mitochondrial ROS release and, presumably, being controlled by high membrane potential, as proposed in the canonical model of \"mild uncoupling\". Our study further promotes a conserved role for UCP1 in the prevention of oxidative stress, which was presumably established during evolution before UCP1 was physiologically integrated into nonshivering thermogenesis.

Ca(2+) -independent phospholipase A2 (iPLA2 ) is hypothesized to control mitochondrial reactive oxygen species (ROS) generation. Here, we modulated the influence of iPLA2 -induced liberation of non-esterified free fatty acids on ROS generation associated with the electron transport chain. We demonstrate enzymatic activity of membrane-associated iPLA2 in native, energized rat brain mitochondria (RBM). Theoretically, enhanced liberation of free fatty acids by iPLA2 modulates mitochondrial ROS generation, either attenuating the reversed electron transport (RET) or deregulating the forward electron transport of electron transport chain. For mimicking such conditions, we probed the effect of docosahexaenoic acid (DHA), a major iPLA2 product on ROS generation. We demonstrate that the adenine nucleotide translocase partly mediates DHA-induced uncoupling, and that low micromolar DHA concentrations diminish RET-dependent ROS generation. Uncoupling proteins have no effect, but the adenine nucleotide translocase inhibitor carboxyatractyloside attenuates DHA-linked uncoupling effect on RET-dependent ROS generation. Under physiological conditions of forward electron transport, low micromolar DHA stimulates ROS generation. Finally, exposure of RBM to the iPLA2 inhibitor bromoenol lactone (BEL) enhanced ROS generation. BEL diminished RBM glutathione content. BEL-treated RBM exhibits reduced Ca(2+) retention capacity and partial depolarization. Thus, we rebut the view that iPLA2 attenuates oxidative stress in brain mitochondria. However, the iPLA2 inhibitor BEL has detrimental activities on energy-dependent mitochondrial functions. The Ca(2+) -independent phospholipase A2 (iPLA2 ), a FFA (free fatty acids)-generating membrane-attached mitochondrial phospholipase, is potential to regulate ROS (reactive oxygen species) generation by mitochondria. FFA can either decrease reversed electron transport (RET)-linked or enhance forward electron transport (FET)-linked ROS generation. In the physiological mode of FET, iPLA2 activity increases ROS generation. The iPLA2 inhibitor BEL exerts detrimental effects on energy-dependent mitochondrial functions.

The Redox-Optimized ROS Balance [R-ORB] hypothesis postulates that the redox environment [RE] is the main intermediary between mitochondrial respiration and reactive oxygen species [ROS]. According to R-ORB, ROS emission levels will attain a minimum vs. RE when respiratory rate (VO2) reaches a maximum following ADP stimulation, a tenet that we test herein in isolated heart mitochondria under forward electron transport [FET]. ROS emission increased two-fold as a function of changes in the RE (~400 to ~900mV·mM) in state 4 respiration elicited by increasing glutamate/malate (G/M). In G/M energized mitochondria, ROS emission decreases two-fold for RE ~500 to ~300mV·mM in state 3 respiration at increasing ADP. Stressed mitochondria released higher ROS, that was only weakly dependent on RE under state 3. As a function of VO2, the ROS dependence on RE was strong between ~550 and ~350mV·mM, when VO2 is maximal, primarily due to changes in glutathione redox potential. A similar dependence was observed with stressed mitochondria, but over a significantly more oxidized RE and ~3-fold higher ROS emission overall, as compared with non-stressed controls. We conclude that under non-stressful conditions mitochondrial ROS efflux decreases when the RE becomes less reduced within a range in which VO2 is maximal. These results agree with the R-ORB postulate that mitochondria minimize ROS emission as they maximize VO2 and ATP synthesis. This relationship is altered quantitatively, but not qualitatively, by oxidative stress although stressed mitochondria exhibit diminished energetic performance and increased ROS release.