Mussel and mammalian ATP synthase share the same bioenergetic cost of ATP

The molecular mechanism by which the membrane-embedded F_O sector of the mitochondrial ATP synthase translocates protons, thus dissipating the transmembrane protonmotive force and leading to ATP synthesis, involves the neutralization of the carboxylate residues of the c-ring. Carboxylates are thought to constitute the binding sites for ion translocation. In order to cast light on this mechanism, we exploited N,N’-dicyclohexylcarbodiimide, which covalently binds to F_O c-ring carboxylates, and ionophores which selectively modulate the transmembrane electric (Δφ) and chemical (ΔpH) gradients such as valinomycin, nigericin and dinitrophenol. ATP hydrolysis was evaluated in mitochondrial preparations and/or inside-out submitochondrial particles from mussel and mammalian tissues under different experimental conditions. The experiments pointed out striking similarities between mussel and mammalian mitochondrial ATP synthase. Our results support the hypothesis that the ATP synthase of Mytilus galloprovincialis induces intersubunit torque generation and translocates H⁺ by coordinating the hydronium ion (H₃O⁺) in the ion binding site of F_O. Our results are consistent with the hypothesis that in mussel mitochondria the main component of the electrochemical gradient driving proton flux and ATP synthesis is Δφ. Therefore, mussel F_O probably contains a small c-ring, which implies a low bioenergetic cost of making ATP as in mammals. These features which make mussel mitochondria as efficient in ATP production as mammalian ones may be especially advantageous in facultative aerobic species which intermittently exploit mitochondrial respiration to generate ATP.     

Metabolic pathways coupled to H+ transport in turtle urinary bladder

Summary Active H⁺ transport in the turtle urinary bladder is mediated by an ATPase. Although the source of ATP is usually mitochondrial oxidative phosphorylation, it is possible because of intracellular compartmentalization or cellular heterogeneity that one metabolic pathway exclusively provides ATP to the pump. To examine this we performed several types of experiments. In one, the coupling between the rate of transport and the rate of oxidation of¹⁴C-labeled substrates was studied. We found that there was coupling between H⁺ transport and glucose, butyrate, oleate, and -OH-butyrate oxidation. In another set of experiments we depleted turtle bladders of their endogenous substrates and tested the effect of a number of substrates on the rate of transport. We found that glucose, pyruvate, lactate, actetate, butyrate and -OH butyrate all stimulated H⁺ transport. In a third set of experiments we found no coupling between H⁺ transport and lactate production. Finally, we found that reduction of H⁺ transport by mucosal acidification resulted in an increase in epithelial cell ATP concentrations and a decrease in ADP levels.These results suggest that the H⁺ pump receives its ATP from carbohydrate and fatty acid oxidation. The changes in ATP and ADP levels provide an initial explanation for the coupling of H⁺ transport to the rate of cellular oxidative metabolism.     

Ca2+ binding to F‐ATP synthase β subunit triggers the mitochondrial permeability transition

F‐ATP synthases convert the electrochemical energy of the H⁺ gradient into the chemical energy of ATP with remarkable efficiency. Mitochondrial F‐ATP synthases can also undergo a Ca²⁺‐dependent transformation to form channels with properties matching those of the permeability transition pore (PTP), a key player in cell death. The Ca²⁺ binding site and the mechanism(s) through which Ca²⁺ can transform the energy‐conserving enzyme into a dissipative structure promoting cell death remain unknown. Through in vitro, in vivo and in silico studies we (i) pinpoint the “Ca²⁺‐trigger site” of the PTP to the catalytic site of the F‐ATP synthase β subunit and (ii) define a conformational change that propagates from the catalytic site through OSCP and the lateral stalk to the inner membrane. T163S mutants of the β subunit, which show a selective decrease in Ca²⁺‐ATP hydrolysis, confer resistance to Ca²⁺‐induced, PTP‐dependent death in cells and developing zebrafish embryos. These findings are a major advance in the molecular definition of the transition of F‐ATP synthase to a channel and of its role in cell death.     

In vivo inhibition of the mitochondrial H+‐ATP synthase in neurons promotes metabolic preconditioning

A key transducer in energy conservation and signaling cell death is the mitochondrial H⁺‐ATP synthase. The expression of the ATPase inhibitory factor 1 (IF1) is a strategy used by cancer cells to inhibit the activity of the H⁺‐ATP synthase to generate a ROS signal that switches on cellular programs of survival. We have generated a mouse model expressing a mutant of human IF1 in brain neurons to assess the role of the H⁺‐ATP synthase in cell death in vivo. The expression of hIF1 inhibits the activity of oxidative phosphorylation and mediates the shift of neurons to an enhanced aerobic glycolysis. Metabolic reprogramming induces brain preconditioning affording protection against quinolinic acid‐induced excitotoxicity. Mechanistically, preconditioning involves the activation of the Akt/p70S6K and PARP repair pathways and Bcl‐xL protection from cell death. Overall, our findings provide the first in vivo evidence highlighting the H⁺‐ATP synthase as a target to prevent neuronal cell death.     

Iron transport by proteoliposomes containing mitochondrial F1Fo ATP synthase isolated from rat heart

In this work, we present evidence of Fe²⁺ transport by rat heart mitochondrial F₁F_o ATP synthase. Iron uptake by the vesicles containing the enzyme was concentration- and temperature-dependent, with an optimum temperature of 37 °C. Both ATP and ADP stimulated iron uptake in a concentration-dependent manner, whereas AMP, AMPPCP, and mADP did not. Inhibitors of the enzyme, oligomycin, and resveratrol similarly blocked iron transport. The iron uptake was confirmed by inhibition using specific antibodies against the α, β, and c subunits of the enzyme. Interestingly, slight transport of common divalent and trivalent metal ions such as Mg⁺², Ca⁺², Mn⁺², Zn⁺², Cu⁺², Fe⁺³, and Al⁺³ was observed. Moreover, Cu⁺², even in the nM range, inhibited iron uptake and attained maximum inhibition of approximately 56%. Inorganic phosphate (Pi) in the medium exerted an opposite effect depending on the type of adenosine nucleotide, which was suppressed with ATP, but enhanced with ADP. A similarly stimulating effect of ATP and ADP with an inverse effect of Pi suggests that the activity of ATPase and ATP synthase may be associated with iron uptake in a different manner, probably via antiport of H⁺.     

α-Synuclein and mitochondrial bioenergetics regulate tetrahydrobiopterin levels in a human dopaminergic model of Parkinson disease

Parkinson disease (PD) is a multifactorial disease resulting in preferential death of the dopaminergic neurons in the substantia nigra. Studies of PD-linked genes and toxin-induced models of PD have implicated mitochondrial dysfunction, oxidative stress, and the misfolding and aggregation of α-synuclein (α-syn) as key factors in disease initiation and progression. Many of these features of PD may be modeled in cells or animal models using the neurotoxin 1-methyl-4-phenylpyridinium (MPP⁺). Reducing oxidative stress and nitric oxide synthase (NOS) activity has been shown to be protective in cell or animal models of MPP⁺ toxicity. We have previously demonstrated that siRNA-mediated knockdown of α-syn lowers the activity of both dopamine transporter and NOS activity and protects dopaminergic neuron-like cells from MPP⁺ toxicity. Here, we demonstrate that α-syn knockdown and modulators of oxidative stress/NOS activation protect cells from MPP⁺-induced toxicity via postmitochondrial mechanisms rather than by a rescue of the decrease in mitochondrial oxidative phosphorylation caused by MPP⁺ exposure. We demonstrate that MPP⁺ significantly decreases the synthesis of the antioxidant and obligate cofactor of NOS and TH tetrahydrobiopterin (BH4) through decreased cellular GTP/ATP levels. Furthermore, we demonstrate that RNAi knockdown of α-syn results in a nearly twofold increase in GTP cyclohydrolase I activity and a concomitant increase in basal BH4 levels. Together, these results demonstrate that both mitochondrial activity and α-syn play roles in modulating cellular BH4 levels.     

Kinetics and Energetics of Light-enhanced Potassium Absorption by Corn Leaf Tissue

The effect of illumination on the absorption of K⁺ by leaf tissue of Zea mays was investigated. The rate of K⁺ absorption was enhanced by exposure of slices of corn leaf tissue to light, even of relatively low intensities. Potassium was transported inward, with virtually no efflux of previously accumulated K⁺. The evidence indicates that the transport mechanism for absorption of K⁺ is the same in the light as in the dark, but that the source of energy for absorption of K⁺ is different in the light from that in the dark. Various anti-metabolites were used to establish that the energy utilized for active ion transport in the light came partly from ATP supplied by cyclic photophosphorylation. Expenditure of ATP was required in the dark too, but this ATP was formed by oxidative phosphorylation. Establishing the ultimate source of energy for active ion uptake by higher plants might be facilitated by demonstration of an ion-transport process that is not linked directly with the transfer of electrons in the mitochondrial cytochrome chain.     

Stanniocalcin-1 Controls Ion Regulation Functions of Ion-transporting Epithelium Other than Calcium Balance

Stanniocalcin-1 (STC-1) was first identified to involve in Ca²⁺ homeostasis in teleosts, and was thought to act as a hypocalcemic hormone in vertebrate. Recent studies suggested that STC-1 exhibits broad effects on ion balance, not confines to Ca²⁺, but the mechanism of this regulation process remains largely unknown. Here, we used zebrafish embryos as an alternative in vivo model to investigate how STC-1 regulates transepithelial ion transport function in ion-transporting epithelium. Expression of stc-1 mRNA in zebrafish embryos was increased in high-Ca²⁺ environments but decreased by acidic and ion-deficient treatments while overexpression of stc-1 impaired the hypotonic acclimation by decreasing whole body Ca²⁺, Na⁺, and Cl^- contents and H⁺ secretion ability. Injection of STC-1 mRNA also down-regulated mRNA expressions of epithelial Ca²⁺ channel, H⁺-ATPase, and Na⁺-Cl^- cotransporter, suggesting the roles of STC-1 in regulation of ions other than Ca²⁺. Knockdown of STC-1 caused an increase in ionocyte progenitors (foxi3a as the marker) and mature ionocytes (ion transporters as the markers), but did not affect epithelium stem cells (p63 as the marker) in the embryonic skin. Overexpression of STC-1 had the corresponding opposite effect on ionocyte progenitors, mature ionocytes in the embryonic skin. Taken together, STC-1 negatively regulates the number of ionocytes to reduce ionocyte functions. This process is important for body fluid ionic homeostasis, which is achieved by the regulation of ion transport functions in ionocytes. The present findings provide new insights into the broader functions of STC-1, a hypocalcemic hormone.     

ATP4A gene regulatory network for fine-tuning of proton pump and ion channels

The ATP4A encodes α subunit of H⁺, K⁺-ATPase that contains catalytic sites of the enzyme forming pores through cell membrane which allows the ion transport. H⁺, K⁺-ATPase is a membrane bound P-type ATPase enzyme which is found on the surface of parietal cells and uses the energy derived from each cycle of ATP hydrolysis that can help in exchanging ions (H⁺, K⁺ and Cl^?) across the cell membrane secreting acid into the gastric lumen. The 3-D model of α-subunit of H⁺, K⁺-ATPase was generated by homology modeling. It was evaluated and validated on the basis of free energies and amino acid residues. The inhibitor binding amino acid active pockets were identified in the 3-D model by molecular docking. The two drugs Omeprazole and Rabeprazole were found more potent interactions with generated model of α-subunit of H⁺, K⁺-ATPase on the basis of their affinity between drug–protein interactions. We have generated ATP4A gene regulatory networks for interactions with other proteins which involved in regulation that can help in fine-tuning of proton pump and ion channels. These findings provide a new dimension for discovery and development of proton pump inhibitors and gene regulation of the ATPase. It can be helpful in better understanding of human physiology and also using synthetic biology strategy for reprogramming of parietal cells for control of gastric ulcers.     

ATP synthases from archaea: The beauty of a molecular motor

Archaea live under different environmental conditions, such as high salinity, extreme pHs and cold or hot temperatures. How energy is conserved under such harsh environmental conditions is a major question in cellular bioenergetics of archaea. The key enzymes in energy conservation are the archaeal A₁A_O ATP synthases, a class of ATP synthases distinct from the F₁F_O ATP synthase ATP synthase found in bacteria, mitochondria and chloroplasts and the V₁V_O ATPases of eukaryotes. A₁A_O ATP synthases have distinct structural features such as a collar-like structure, an extended central stalk, and two peripheral stalks possibly stabilizing the A₁A_O ATP synthase during rotation in ATP synthesis/hydrolysis at high temperatures as well as to provide the storage of transient elastic energy during ion-pumping and ATP synthesis/-hydrolysis. High resolution structures of individual subunits and subcomplexes have been obtained in recent years that shed new light on the function and mechanism of this unique class of ATP synthases. An outstanding feature of archaeal A₁A_O ATP synthases is their diversity in size of rotor subunits and the coupling ion used for ATP synthesis with H⁺, Na⁺ or even H⁺ and Na⁺ using enzymes. The evolution of the H⁺ binding site to a Na⁺ binding site and its implications for the energy metabolism and physiology of the cell are discussed.     

Elucidation of antimicrobial activity and mechanism of action by N-substituted carbazole derivatives

Compounds belonging to a carbazole series have been identified as potent fungal plasma membrane proton adenosine triphophatase (H⁺-ATPase) inhibitors with a broad spectrum of antifungal activity. The carbazole compounds inhibit the adenosine triphosphate (ATP) hydrolysis activity of the essential fungal H⁺-ATPase, thereby functionally inhibiting the extrusion of protons and extracellular acidification, processes that are responsible for maintaining high plasma membrane potential. The compound class binds to and inhibits the H⁺-ATPase within minutes, leading to fungal death after 1–3 h of compound exposure in vitro. The tested compounds are not selective for the fungal H⁺-ATPase, exhibiting an overlap of inhibitory activity with the mammalian protein family of P-type ATPases; the sarco(endo)plasmic reticulum calcium ATPase (Ca²⁺-ATPase) and the sodium potassium ATPase (Na⁺,K⁺-ATPase). The ion transport in the P-type ATPases is energized by the conversion of ATP to adenosine diphosphate (ADP) and phosphate and a general inhibitory mechanism mediated by the carbazole derivative could therefore be blocking of the active site. However, biochemical studies show that increased concentrations of ATP do not change the inhibitory activity of the carbazoles suggesting they act as allosteric inhibitors. Furthermore decreased levels of intracellular ATP would suggest that the compounds inhibit the H⁺-ATPase indirectly, but Candida albicans cells exposed to potent H⁺-ATPase-inhibitory carbazoles result in increased levels of intracellular ATP, indicating direct inhibition of H⁺-ATPase.     

Up-regulation of the ATPase Inhibitory Factor 1 (IF1) of the Mitochondrial H+-ATP Synthase in Human Tumors Mediates the Metabolic Shift of Cancer Cells to a Warburg Phenotype

The H⁺-ATP synthase is a reversible engine of mitochondria that synthesizes or hydrolyzes ATP upon changes in cell physiology. ATP synthase dysfunction is involved in the onset and progression of diverse human pathologies. During ischemia, the ATP hydrolytic activity of the enzyme is inhibited by the ATPase inhibitory factor 1 (IF1). The expression of IF1 in human tissues and its participation in the development of human pathology are unknown. Here, we have developed monoclonal antibodies against human IF1 and determined its expression in paired normal and tumor biopsies of human carcinomas. We show that the relative mitochondrial content of IF1 increases significantly in carcinomas, suggesting the participation of IF1 in oncogenesis. The expression of IF1 varies significantly in cancer cell lines. To investigate the functional activity of IF1 in cancer, we have manipulated its cellular content. Overexpression of IF1 or of its pH-insensitive H49K mutant in cells that express low levels of IF1 triggers the up-regulation of aerobic glycolysis and the inhibition of oxidative phosphorylation with concurrent mitochondrial hyperpolarization. Treatment of the cells with the H⁺-ATP synthase inhibitor oligomycin mimicked the effects of IF1 overexpression. Conversely, small interfering RNA-mediated silencing of IF1 in cells that express high levels of IF1 promotes the down-regulation of aerobic glycolysis and the increase in oxidative phosphorylation. Overall, these findings support that the mitochondrial content of IF1 controls the activity of oxidative phosphorylation mediating the shift of cancer cells to an enhanced aerobic glycolysis, thus supporting an oncogenic role for the de-regulated expression of IF1 in cancer.     

Effects of ethacrynic acid on ion transport and energy metabolism in slices of avian salt gland and of mammalian liver and kidney cortex

Summary Ethacrynic acid greatly inhibited net transport of ions and aerobic, energyconserving metabolism in slices of avian salt gland, rat liver, and rat and guinea-pig kidney cortex. The effects of increasing concentrations of ethacrynic acid on the transport of Na⁺, K⁺ and Cl^– ran closely parallel to its effects on tissue ATP levels and respiration. The concentration needed for maximal inhibition of transport reduced ATP levels by 80–90%. Respiration was reduced by 80–90% in salt gland and kidney cortex, and by a maximum of 30% in liver slices. The effects of low concentrations of ethacrynic acid required time to become fully manifest in some tissues, and the development of transport inhibition followed a similar course to decline of respiration and ATP levels. Ca²⁺ extrusion by liver cells was inhibited by ethacrynic acid. The concentration dependence of the inhibition was similar to that shown by the other transport systems inhibited. There was no distinction evident between the sensitivity of Na⁺ extrusion and of K⁺ accumulation to the diuretic. Lactate production increased as respiration decreased in the presence of increasing concentrations of ethacrynic acid. We conclude that ethacrynic acid acted primarily as an inhibitor of mitochondrial respiration and ATP synthesis in the tissue slices, and that inhibition of ion transport was a nonspecific consequence of the failure of the energy supply.     

Effect of pH and Ca2+ on the retention of Ca2+ by rat liver mitochondria.

A transient Ca²⁺ release from preloaded mitochondria can be induced by a sudden decrease in the pH of the outer medium from 8.0 or 7.4 to 6.8. In the presence of inorganic phosphate the released Ca²⁺ is not taken up again. Upon Ca²⁺ addition to respiring mitochondria the mitochondrial membrane potential (Δ♀) decreases to a new resting level. A further decrease in Δ♀ occurs after the decrease in pH from 7.4 to 6.8, concomitant with the reuptake phase of the Ca²⁺ release. Phosphate, EGTA, and ruthenium red restore Δ♀ to its initial level. If phosphate is present initially, only transient changes in Δ♀ occur upon addition of Ca²⁺ or H⁺ ions. Only a small transient change in Δ♀ upon H⁺ ion addition is seen in the absence of accumulated Ca²⁺. La³⁺, a competitive inhibitor of Ca²⁺ transport, prevents the H⁺ ion-induced Ca²⁺ efflux, whereas this is not the case in the presence of the noncompetitive inhibitor ruthenium red. Ruthenium red, however, prevents the reuptake phase. Mg²⁺, an inhibitor of the surface binding of Ca²⁺, has no or only a slight effect on the H⁺ ion-induced Ca²⁺ release. Mitochondria preloaded with Ca²⁺ release a small fraction of Ca²⁺ during the subsequent uptake of another pulse of Ca²⁺. The results indicate that at least one pool of mitochondrial Ca²⁺ exists in a mobile state. The possible existence of a $\text{H}{}^{\mathrm{+}}\text{Ca}{}^{\mathrm{2+}}$ exchanger in the mitochondrial membrane is discussed.     

Na+ Transport by the A1AO-ATP Synthase Purified from Thermococcus onnurineus and Reconstituted into Liposomes

The ATP synthase of many archaea has the conserved sodium ion binding motif in its rotor subunit, implying that these A₁A_O-ATP synthases use Na⁺ as coupling ion. However, this has never been experimentally verified with a purified system. To experimentally address the nature of the coupling ion, we have purified the A₁A_O-ATP synthase from T. onnurineus. It contains nine subunits that are functionally coupled. The enzyme hydrolyzed ATP, CTP, GTP, UTP, and ITP with nearly identical activities of around 40 units/mg of protein and was active over a wide pH range with maximal activity at pH 7. Noteworthy was the temperature profile. ATP hydrolysis was maximal at 80 °C and still retained an activity of 2.5 units/mg of protein at 45 °C. The high activity of the enzyme at 45 °C opened, for the first time, a way to directly measure ion transport in an A₁A_O-ATP synthase. Therefore, the enzyme was reconstituted into liposomes generated from Escherichia coli lipids. These proteoliposomes were still active at 45 °C and coupled ATP hydrolysis to primary and electrogenic Na⁺ transport. This is the first proof of Na⁺ transport by an A₁A_O-ATP synthase and these findings are discussed in light of the distribution of the sodium ion binding motif in archaea and the role of Na⁺ in the bioenergetics of archaea.     

Drosophila Sirt2/mammalian SIRT3 deacetylates ATP synthase β and regulates complex V activity

Adenosine triphosphate (ATP) synthase β, the catalytic subunit of mitochondrial complex V, synthesizes ATP. We show that ATP synthase β is deacetylated by a human nicotinamide adenine dinucleotide (NAD⁺)–dependent protein deacetylase, sirtuin 3, and its Drosophila melanogaster homologue, dSirt2. dsirt2 mutant flies displayed increased acetylation of specific Lys residues in ATP synthase β and decreased complex V activity. Overexpression of dSirt2 increased complex V activity. Substitution of Lys 259 and Lys 480 with Arg in human ATP synthase β, mimicking deacetylation, increased complex V activity, whereas substitution with Gln, mimicking acetylation, decreased activity. Mass spectrometry and proteomic experiments from wild-type and dsirt2 mitochondria identified the Drosophila mitochondrial acetylome and revealed dSirt2 as an important regulator of mitochondrial energy metabolism. Additionally, we unravel a ceramide–NAD⁺–sirtuin axis wherein increased ceramide, a sphingolipid known to induce stress responses, resulted in depletion of NAD⁺ and consequent decrease in sirtuin activity. These results provide insight into sirtuin-mediated regulation of complex V and reveal a novel link between ceramide and Drosophila acetylome.     

Erythrosin B as an effective inhibitor of electrogenic H+ extrusion

Abstract In 24-h-genninaled radish seedlings erythrosin B (EB), an effective inhibitor of microsomal as well as of partially purified vanadate-sensitive ATPase markedly inhibited the basal and the FC-stimulated proton extrusion, and induced a rapid depolarization of FC-hyperpolarized trans-membrane electric potential (PD) without causing any significant change of ATP level. The effects of EB on H⁺ extrusion were partially additive with those of vanadatc, another inhibitor of plasma membrane H⁺-ATPase. These effects are interpreted as due to a direct inhibition by EB on plasma membrane H⁺-ATPase involved in H⁺ electrogenic transport in the higher plants.     

ATM-mediated mitochondrial damage response triggered by nuclear DNA damage in normal human lung fibroblasts

Ionizing radiation (IR) elevates mitochondrial oxidative phosphorylation (OXPHOS) in response to the energy requirement for DNA damage responses. Reactive oxygen species (ROS) released during mitochondrial OXPHOS may cause oxidative damage to mitochondria in irradiated cells. In this paper, we investigated the association between nuclear DNA damage and mitochondrial damage following IR in normal human lung fibroblasts. In contrast to low-doses of acute single radiation, continuous exposure of chronic radiation or long-term exposure of fractionated radiation (FR) induced persistent Rad51 and γ-H2AX foci at least 24 hours after IR in irradiated cells. Additionally, long-term FR increased mitochondrial ROS accompanied with enhanced mitochondrial membrane potential (ΔΨm) and mitochondrial complex IV (cytochrome c oxidase) activity. Mitochondrial ROS released from the respiratory chain complex I caused oxidative damage to mitochondria. Inhibition of ATM kinase or ATM loss eliminated nuclear DNA damage recognition and mitochondrial radiation responses. Consequently, nuclear DNA damage activates ATM which in turn increases ROS level and subsequently induces mitochondrial damage in irradiated cells.

In conclusion, we demonstrated that ATM is essential in the mitochondrial radiation responses in irradiated cells. We further demonstrated that ATM is involved in signal transduction from nucleus to the mitochondria in response to IR.