Cytosolic ATP Research Articles

Mitochondrial respiration is essential for photosynthesis-dependent ATP supply of the plant cytosol.

Plants rely on solar energy to synthesize ATP and NADPH for photosynthetic carbon fixation and all cellular need. Mitochondrial respiration is essential in plants, but this may be due to heterotrophic bottlenecks during plant development or because it is also necessary in photosynthetically active cells. In this study, we examined in vivo changes of cytosolic ATP concentration in response to light, employing a biosensing strategy in the moss Physcomitrium patens and revealing increased cytosolic ATP concentration caused by photosynthetic activity. Plants depleted of respiratory Complex I showed decreased cytosolic ATP accumulation, highlighting a critical role of mitochondrial respiration in light-dependent ATP supply of the cytosol. Consistently, targeting mitochondrial ATP production directly, through the construction of mutants deficient in mitochondrial ATPase (complex V), led to drastic growth reduction, despite only minor alterations in photosynthetic electron transport activity. Since P. patens is photoautotrophic throughout its development, we conclude that heterotrophic bottlenecks cannot account for the indispensable role of mitochondrial respiration in plants. Instead, our results support that mitochondrial respiration is essential for ATP provision to the cytosol in photosynthesizing cells. Mitochondrial respiration provides metabolic integration, ensuring supply of cytosolic ATP essential for supporting plant growth and development.

Deletion of murine astrocytic vesicular nucleotide transporter increases anxiety and depressive-like behavior and attenuates motivation for reward.

Astrocytes are multi-functional glial cells in the central nervous system that play critical roles in modulation of metabolism, extracellular ion and neurotransmitter levels, and synaptic plasticity. Astrocyte-derived signaling molecules mediate many of these modulatory functions of astrocytes, including vesicular release of ATP. In the present study, we used a unique genetic mouse model to investigate the functional significance of astrocytic exocytosis of ATP. Using primary cultured astrocytes, we show that loss of vesicular nucleotide transporter (Vnut), a primary transporter responsible for loading cytosolic ATP into the secretory vesicles, dramatically reduces ATP loading into secretory lysosomes and ATP release, without any change in the molecular machinery of exocytosis or total intracellular ATP content. Deletion of astrocytic Vnut in adult mice leads to increased anxiety, depressive-like behaviors, and decreased motivation for reward, especially in females, without significant impact on food intake, systemic glucose metabolism, cognition, or sociability. These behavioral alterations are associated with significant decreases in the basal extracellular dopamine levels in the nucleus accumbens. Likewise, ex vivo brain slices from these mice show a strong trend toward a reduction in evoked dopamine release in the nucleus accumbens. Mechanistically, the reduced dopamine signaling we observed is likely due to an increased expression of monoamine oxidases. Together, these data demonstrate a key modulatory role of astrocytic exocytosis of ATP in anxiety, depressive-like behavior, and motivation for reward, by regulating the mesolimbic dopamine circuitry.

ATP as a signaling molecule

The review considers the effects of extracellular ATP mediated by plasma membrane purinoreceptors in the cells of different tissues, in particular, myometrium. Recently published results suggest that cytosolic ATP may also play a role of signaling molecule, as indicated by the detection of the ATP receptor not only in the plasma membrane, but also in mitochondria. The authors have shown that ionized Ca2+ concentration in the rat myometrium mitochondria matrix is regulated by ATP at the absence of exogenous Ca2+. ATP concentration-dependent increase of [Ca2+]m was not affected in the presence of the mitochondrial Ca2+-uniporter blocker ruthenium red, the mitochondrial pore blocker cyclosporine A, or ATP synthase inhibitor oligomycin. It is assumed that cytosolic ATP could be a signaling molecule that regulates at least the Ca2+ ions exchange in mitochondria. Keywords: Ca2+ ions exchange, extracellular and cytosolic ATP, mitochondria, P2X7 receptors

AGC1-mediated Metabolic Reprogramming and Autophagy Sustain Survival of Hepatocellular Carcinoma Cells under Glutamine Deprivation.

The dependence of hepatocellular carcinoma (HCC) cells on glutamine suggests the feasibility of targeting glutamine metabolism for therapy. However, drugs inhibiting glutamine uptake and breakdown have not shown promising outcomes. Therefore, investigating the mechanism of glutamine metabolism reprogramming in HCC cells is crucial. We used bioinformatics approaches to investigate the metabolic flux of glutamine in HCC cells and validated it using qRT-PCR and western blotting. HCC cells were cultured in glutamine-deprived medium, and changes in glutamate and ATP levels were monitored. Western blotting was employed to assess the expression of AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) and autophagy-related proteins. The impact of Solute carrier family 25 member 12 (AGC1) on HCC cell proliferation was studied using CCK-8 and colony formation assays. Furthermore, the effects of AGC1 knockdown via siRNA on metabolic reprogramming and energy supply during glutamine deprivation in HCC were explored. During glutamine deprivation, HCC cells sustain cytosolic asparagine synthesis and ATP production through AGC1. Low ATP levels activate AMPK and inhibit mTOR activation, inducing autophagy to rescue HCC cell survival. Knockdown of AGC1 inhibits mitochondrial aspartate output and continuously activates autophagy, rendering HCC cells more sensitive to glutamine deprivation. AGC1 serves as a critical node in the reprogramming of glutamine metabolism and energy supply in HCC cells. This study provides theoretical support for overcoming resistance to drugs targeting glutamine metabolism.

The mitochondrial orf117Sha gene desynchronizes pollen development and causes pollen abortion in Arabidopsis Sha cytoplasmic male sterility.

Cytoplasmic male sterility (CMS) is of major agronomical relevance in hybrid breeding. In gametophytic CMS, abortion of pollen is determined by the grain genotype, while in sporophytic CMS, it is determined by the mother plant genotype. While several CMS mechanisms have been dissected at the molecular level, gametophytic CMS has not been straightforwardly accessible. We used the gametophytic Sha-CMS in Arabidopsis to characterize the cause and process of pollen abortion by implementing in vivo biosensing in single pollen and mitoTALEN mutagenesis. We obtained conclusive evidence that orf117Sha is the CMS-causing gene, despite distinct characteristics from other CMS genes. We measured the in vivo cytosolic ATP content in single pollen, followed pollen development, and analyzed pollen mitochondrial volume in two genotypes that differed only by the presence of the orf117Sha locus. Our results showed that the Sha-CMS is not triggered by ATP deficiency. Instead, we observed desynchronization of a pollen developmental program. Pollen death occurred independently in pollen grains at diverse stages and was preceded by mitochondrial swelling. We conclude that pollen death is grain-autonomous in Sha-CMS and propose that mitochondrial permeability transition, which was previously described as a hallmark of developmental and environmental-triggered cell death programs, precedes pollen death in Sha-CMS.

Abstract 1046: Miro1 Promotes Mitochondria ER Contact Sites Formation In Vascular Smooth Muscle Cells During G1/s

Vascular smooth muscle cell (VSMC) proliferation plays a key role in vascular disorders including atherosclerosis. Mitochondria promote VSMC proliferation by mitochondrial Ca 2+ regulation, ROS and ATP production. Mitochondria-ER contact sites (MERCS) promote signal transduction to mitochondria. MIRO1, an outer mitochondrial membrane GTPase, drives intracellular mitochondrial mobility. In drosophila, MIRO1 controls MERCS formation. However, it is unknown whether this interaction promotes key cellular eukaryotic phenotypes, such as proliferation. Here, we tested whether MIRO1 promotes VSMC proliferation by modulating MERCS and mitochondrial Ca 2+ entry. MIRO1 deletion decreased neointima formation in vivo, and abolished VSMC proliferation and G1/S transition in vitro. In WT VSMCs, proximity ligation assays (PLA) and split-GFP imaging revealed that MERCS formation increased in G1/S phase along with recruitment of MIRO1. MIRO1 deletion abolished cell cycle-dependent regulation of MERCS while increasing the expression of MERCS proteins GRP75 and IP3R. MIRO1 deletion also blocked increases in baseline mitochondrial [Ca 2+ ] by mitoPericam imaging and decreases in ER [Ca 2+ ] by CEPIA-ER in G1/S phase. Additionally, the mitochondrial membrane potential (TMRM) and mitochondrial respiration significantly increased at G1/S in WT VSMCs but not in MIRO1-/- cells. In WT VSMCs, pyruvate dehydrogenase activity increased G1/S phase while cytosolic ATP levels (ATP sensor) were increased. In MIRO1-/- cells, both parameters did not change. These data demonstrate that MERCS formation and mitochondrial [Ca 2+ ] increases during the cell cycle and is controlled by MIRO1. Furthermore, MIRO1 deletion decreases cellular ATP levels in VSMCs. In conclusion, we propose that MIRO1 controls MERCS and increased mitochondrial [Ca 2+ ] that drives metabolic activity and proliferation.

IDF23-0069 Relationship of High-Altitude Hypoxia and Cytosolic ATP with Liver Fibrosis in MAFLD and DM2

IDF23-0069 Relationship of High-Altitude Hypoxia and Cytosolic ATP with Liver Fibrosis in MAFLD and DM2

Pulsed Radiofrequency 2 Hz Preserves the Dorsal Root Ganglion Neuron Physiological Ca2+ Influx, Cytosolic ATP Level, Δψm, and pERK Compared to 4 Hz: An Insight on the Safety of Pulsed Radiofrequency in Pain Management [Response to Letter

Pulsed Radiofrequency 2 Hz Preserves the Dorsal Root Ganglion Neuron Physiological Ca2+ Influx, Cytosolic ATP Level, Δψm, and pERK Compared to 4 Hz: An Insight on the Safety of Pulsed Radiofrequency in Pain Management [Response to Letter

Genetically Encoded Biosensors Unveil Neuronal Injury Dynamics via Multichromatic ATP and Calcium Imaging.

When a cell sustains damage, it liberates cytosolic ATP, which can serve as an injury signal, affecting neighboring cells. This study presents a methodological approach that employs in vitro axotomy and in vivo laser ablation to simulate cellular injury. Specially tailored biosensors are employed to monitor ATP dynamics and calcium transients in injured cells and their surroundings. To simultaneously visualize extracellular and cytosolic ATP, we developed bicistronic constructs featuring GRABATP1.0 and MaLionR biosensors alongside the calcium sensor RCaMP, enabling multiparametric imaging. In addition to transducing primary neuron cultures, we developed another method where we cocultured dorsal root ganglion neurons together with specialized "sniffer" cell lines expressing the bicistronic biosensors. Exploiting these approaches, we successfully demonstrated the release of ATP from the injured neurons and its extracellular diffusion in response to cellular injury in vitro and in vivo. Axotomy triggered intracellular calcium mobilization not only in the injured neuron but also in the intact neighboring cells, providing new insights into ATP's role as an injury signal. The tools developed in this study have demonstrated remarkable efficiency in unraveling the intricacies of ATP-mediated injury signaling.

Mitochondria-derived reactive oxygen species are the likely primary trigger of mitochondrial retrograde signaling in Arabidopsis

Besides their central function in respiration, plant mitochondria play a crucial role in maintaining cellular homeostasis during stress by providing "retrograde" feedback to the nucleus. Despite the growing understanding of this signaling network, the nature of the signals that initiate mitochondrial retrograde regulation (MRR) in plants remains unknown. Here, we investigated the dynamics and causative relationship of a wide range of mitochondria-related parameters for MRR, using a combination of Arabidopsis fluorescent protein biosensor lines, invitro assays, and genetic and pharmacological approaches. We show that previously linked physiological parameters, including changes in cytosolic ATP, NADH/NAD+ ratio, cytosolic reactive oxygen species (ROS), pH, free Ca2+, and mitochondrial membrane potential, may often be correlated with-but are not the primary drivers of-MRR induction in plants. However, we demonstrate that the induced production of mitochondrial ROS is the likely primary trigger for MRR induction in Arabidopsis. Furthermore, we demonstrate that mitochondrial ROS-mediated signaling uses the ER-localized ANAC017-pathway to induce MRR response. Finally, our data suggest that mitochondrially generated ROS can induce MRR without substantially leaking into other cellular compartments such as the cytosol or ER lumen, as previously proposed. Overall, our results offer compelling evidence that mitochondrial ROS elevation is the likely trigger of MRR.

Pulsed Radiofrequency 2 Hz Preserves the Dorsal Root Ganglion Neuron Physiological Ca2+ Influx, Cytosolic ATP Level, Δψm, and pERK Compared to 4 Hz: An Insight on the Safety of Pulsed Radiofrequency in Pain Management [Letter

Pulsed Radiofrequency 2 Hz Preserves the Dorsal Root Ganglion Neuron Physiological Ca2+ Influx, Cytosolic ATP Level, Δψm, and pERK Compared to 4 Hz: An Insight on the Safety of Pulsed Radiofrequency in Pain Management [Letter

A fast method to distinguish between fermentative and respiratory metabolisms in single yeast cells

Saccharomyces cerevisiae adjusts its metabolism based on nutrient availability, typically transitioning from glucose fermentation to ethanol respiration as glucose becomes limiting. However, our understanding of the regulation of metabolism is largely based on population averages, whereas nutrient transitions may cause heterogeneous responses. Here we introduce iCRAFT, a method that couples the ATP Förster resonance energy transfer (FRET)-based biosensor yAT1.03 with Antimycin A to differentiate fermentative and respiratory metabolisms in individual yeast cells. Upon Antimycin A addition, respiratory cells experienced a sharp decrease of the normalized FRET ratio, while respiro-fermentative cells showed no response. Next, we tracked changes in metabolism during the diauxic shift of a glucose pre-grown culture. Following glucose exhaustion, the entire cell population experienced a progressive rise in cytosolic ATP produced via respiration, suggesting a gradual increase in respiratory capacity. Overall, iCRAFT is a robust tool to distinguish fermentation from respiration, offering a new single-cell opportunity to study yeast metabolism.

Mild excitotoxic effect on mitochondrial membrane potential, Ca2+ homeostasis and energy production in a mouse model of Alzheimer’s disease

AbstractBackgroundNeurons are bound to maintain a tight control over their cellular homeostasis in order to preserve their many functions, and specifically to precisely regulate ion fluxes for the generation of action potential and neurotransmission. In particular, the energetic balance has to be finely adjusted since its alteration may hamper cellular functionality with consequent cell suffering and death. Mitochondria play a central role in the homeostatic regulation within the cells due to the many vital tasks they perform in terms of energy production, participation in the regulation of Ca2+ concentration, modulation of intracellular signaling that allow the cell to respond to external solicitationMethodPrimary hippocampal neuronal cultures of 14‐17 DIV from WT and AD mouse model were challenged with μM concentration of glutamate to induce a mild excitotoxic effect; mitochondrial membrane potential, mitochondrial an cytosolic Ca2+ transients and cytosolic ATP levels were evaluated with fluorescent probes. Cells were treated also with inhibitors of the permeability transition pore to evaluate its effect on cell functionalityResultWe observed that in a model of Familial Alzheimer’s disease, hippocampal neurons are sensitive to a modest excitotoxic signal that produces a transient cytosolic and mitochondrial matrix Ca2+ with consequent partial loss of mitochondrial membrane potential and delayed recovery of mitochondrial matrix [Ca2+] which might contribute in the activation of the mitochondria permeability transition pore and consequent impairment of mitochondrial and cellular functions. This is accompanied by a reduced cytosolic ATP content that can result in an energy deficit for the cell. A brief treatment with inhibitors of the permeability transition pore is able to reduce these effects and might have protective roles against exogenous insults to the cells and help mitigate the neuronal loss characteristic of Alzheimer’s disease.ConclusionAD hippocampal neurons are more sensitive to a mild excitotoxic insult that affect mitochondrial functionality and in particular the activation of the permeability transition pore (PTP). Treatment with μM concentrations of two different inhibitors of the PTP prevent mitochondrial impairment.

Postcontraction [acetylcarnitine] reflects interindividual variation in skeletal muscle ATP production patterns in vivo.

In addition to its role in substrate selection (carbohydrate vs. fat) for oxidative metabolism in muscle, acetylcarnitine production may be an important modulator of the energetic pathway by which ATP is produced. A combination of noninvasive magnetic resonance spectroscopy measures of cytosolic acetylcarnitine and ATP production pathways was used to investigate the link between [acetylcarnitine] and energy production in vivo. Intracellular metabolites were measured in the vastus lateralis muscle of eight males (mean: 28.4 yr, range: 25-35) during 8 min of incremental, dynamic contractions (0.5 Hz, 2-min stages at 6%, 9%, 12%, and 15% maximal torque) that increased [acetylcarnitine] approximately fivefold from resting levels. ATP production via oxidative phosphorylation, glycolysis, and the creatine kinase reaction was calculated based on phosphorus metabolites and pH. Spearman rank correlations indicated that postcontraction [acetylcarnitine] was positively associated with both absolute (mM) and relative (% total ATP) glycolytic ATP production (rs = 0.95, P = 0.001; rs = 0.93, P = 0.002), and negatively associated with relative (rs = -0.81, P = 0.02) but not absolute (rs = -0.14, P = 0.75) oxidative ATP production. Thus, acetylcarnitine accumulated more when there was a greater reliance on "nonoxidative" glycolysis and a relatively lower contribution from oxidative phosphorylation, reflecting the fate of pyruvate in working skeletal muscle. Furthermore, these data indicate striking interindividual variation in responses to the energy demand of submaximal contractions. Overall, the results of this preliminary study provide novel evidence of the coupling in vivo between ATP production pathways and the carnitine system.NEW & NOTEWORTHY Production of acetylcarnitine from acetyl-CoA and free carnitine may be important for energy pathway regulation in contracting skeletal muscle. Noninvasive magnetic resonance spectroscopy was used to investigate the link between acetylcarnitine and energy production in the vastus lateralis muscle during dynamic contractions (n = 8 individuals). A positive correlation between acetylcarnitine accumulation and "nonoxidative" glycolysis and an inverse relationship with oxidative phosphorylation, provides novel evidence of the coupling between ATP production and the carnitine system in vivo.

Mitochondrial phosphagen kinases support the volatile power demands of motor nerve terminals.

Motor neurons are the longest neurons in the body, with axon terminals separated from the soma by as much as a meter. These terminals are largely autonomous with regard to their bioenergetic metabolism and must burn energy at a high rate to sustain muscle contraction. Here, through computer simulation and drawing on previously published empirical data, we determined that motor neuron terminals in Drosophilalarvae experience highly volatile power demands. It might not be surprising then, that we discovered the mitochondria in the motor neuron terminals of both Drosophilaand mice to be heavily decorated with phosphagen kinases - a key element in an energy storage and buffering system well-characterized in fast-twitch muscle fibres. Knockdown of arginine kinase 1 (ArgK1) in Drosophila larval motor neurons led to several bioenergetic deficits, including mitochondrial matrix acidification and a faster decline in the cytosol ATP to ADP ratio during axon burst firing. KEY POINTS: Neurons commonly fire in bursts imposing highly volatile demands on the bioenergetic machinery that generates ATP. Using a computational approach, we built profiles of presynaptic power demand at the level of single action potentials, as well as the transition from rest to sustained activity. Phosphagen systems are known to buffer ATP levels in muscles and we demonstrate that phosphagen kinases, which support such phosphagen systems, also localize to mitochondria in motor nerve terminals of fruit flies and mice. By knocking down phosphagen kinases in fruit fly motor nerve terminals, and using fluorescent reporters of the ATP:ADP ratio, lactate, pH and Ca2+ , we demonstrate a role for phosphagen kinases in stabilizing presynaptic ATP levels. These data indicate that the maintenance of phosphagen systems in motor neurons, and not just muscle, could be a beneficial initiative in sustaining musculoskeletal health and performance.

Abstract 14250: ND-13, a Small DJ-1 Derived Peptide, Confers Cardioprotection by Targeting Mitochondrial Homeostasis

Objectives: DJ-1 protein is a multifunctional protein with cardioprotective effects against acute myocardial ischemia-reperfusion injury (IRI). This study aims to elucidate whether ND-13, a small peptide derived from DJ-1, can confer acute cardioprotection after IRI by preserving mitochondrial homeostasis through improvements in mitochondrial respiratory capacity and regulating the balance between mitochondrial fusion/fission. By enhancing mitochondrial respiration and maintaining the integrity of the mitochondrial network, ND-13 may provide a novel therapeutic strategy for cardioprotection in cardiomyocytes following IRI. Methods: In vitro , ex vivo and in vivo murine models of IRI were used to elucidate the cardioprotective effects of ND-13. Infarct size, cardiac function and mitochondrial function were assessed. Results: ND-13 was shown to confer cardioprotection in three distinct models of IRI: (i) in vitro , cardiomyocyte (CM) death was reduced by 42% (64±1% untreated vs 37±2% treated; p≤0.01); (ii) ex vivo Langendorrf, with 43% decrease in infarct size (51±6.4% untreated vs 29±8.8% treated; p≤0.05); (iii) in vivo , with 35% reduction in infarct size (49±6.4% untreated vs 32±5.0% treated; p≤0.0001). CMs treated with ND-13 in the in vitro model of IRI demonstrated a significant increase in mitochondrial respiration rates, (basal: untreated 37±3 vs treated 63±10 pmol/min/cells, p≤0.05; maximal: untreated 286±20 vs treated 494±53 pmol/min/cells, p≤0.001; and spare respiratory capacity: untreated 251±18 vs treated 421±43 pmol/min/cells, p≤0.001) and a significant increase in levels of cytosolic ATP (untreated 4.0 ± 0.3 vs treated 5.6 ± 0.5 μM, p≤0.05). Conclusion: We demonstrate for the first time, cardioprotection with the DJ-1-derived peptide, ND-13, and this beneficial effect appears to be mediated by its mitoprotective actions with enhanced mitochondrial respiratory efficiency and increased ATP production. Upcoming research aims to explore if ND-13 promotes oxidative phosphorylation by directly binding to complexes of the electron transport chain to maximize respiratory function or by enhancing pyruvate dehydrogenase activity, thereby potentially influencing the regulation of the fission/fusion process.

Pulsed Radiofrequency 2 Hz Preserves the Dorsal Root Ganglion Neuron Physiological Ca2+ Influx, Cytosolic ATP Level, Δψm, and pERK Compared to 4 Hz: An Insight on the Safety of Pulsed Radiofrequency in Pain Management

Pulsed Radiofrequency 2 Hz Preserves the Dorsal Root Ganglion Neuron Physiological Ca2+ Influx, Cytosolic ATP Level, Δψm, and pERK Compared to 4 Hz: An Insight on the Safety of Pulsed Radiofrequency in Pain Management

Sodium pyruvate improves the plasma amino acid profile in rats with L-arginine-induced acute pancreatitis.

Plasma amino acid levels are altered upon many pathological conditions including acute pancreatitis. It is unclear whether amino acids can be used as specific biomarker of acute pancreatitis severity or recovery. Development of acute pancreatitis is associated with mitochondrial dysfunction and decreased cytosolic ATP level. Sodium pyruvate is considered as a potential treatment of pancreatitis due to its ability to sustain mitochondrial oxidative and ATP-productive capacity in vitro. This study investigated the effect of sodium pyruvate on pancreatic morphology and plasma amino acid levels in rats with acute pancreatitis. Acute pancreatitis in rats was induced by administration of L-arginine (5g/kg) Experimental treatment group received sodium pyruvate (1g/kg) for 4days. On day 8 of the experiment, animals were killed, blood was collected and plasma amino acid concentration was determined with high-performance liquid chromatography. Histological examination showed large areas of fibrosis in the pancreas of animals treated with L-arginine irrespectively of sodium pyruvate administration. Sodium pyruvate improved the plasma amino acid levels. Rats with acute pancreatitis had significantly lower levels of most essential and non-essential amino acids and increased glutamate and aspartate in plasma. Administration of sodium pyruvate completely or partially restored the levels of methionine, phenylalanine, tryptophan, leucine, isoleucine, aspartate, asparagine and ornithine levels, while increasing glutamine and serine to levels significantly higher than control. Plasma lysine, alanine, arginine and taurine remained unaffected in all experimental groups. Sodium pyruvate may be considered for use as a maintenance therapy in acute pancreatitis.

ATP yield of plant respiration: potential, actual and unknown.

The ATP yield of plant respiration (ATP/hexose unit respired) quantitatively links active heterotrophic processes with substrate consumption. Despite its importance, plant respiratory ATP yield is uncertain. The aim here was to integrate current knowledge of cellular mechanisms with inferences required to fill knowledge gaps to generate a contemporary estimate of respiratory ATP yield and identify important unknowns. A numerical balance sheet model combining respiratory carbon metabolism and electron transport pathways with uses of the resulting transmembrane electrochemical proton gradient was created and parameterized for healthy, non-photosynthesizing plant cells catabolizing sucrose or starch to produce cytosolic ATP. Mechanistically, the number of c subunits in the mitochondrial ATP synthase Fo sector c-ring, which is unquantified in plants, affects ATP yield. A value of 10 was (justifiably) used in the model, in which case respiration of sucrose potentially yields about 27.5 ATP/hexose (0.5 ATP/hexose more from starch). Actual ATP yield often will be smaller than its potential due to bypasses of energy-conserving reactions in the respiratory chain, even in unstressed plants. Notably, all else being optimal, if 25 % of respiratory O2 uptake is via the alternative oxidase - a typically observed fraction - ATP yield falls 15 % below its potential. Plant respiratory ATP yield is smaller than often assumed (certainly less than older textbook values of 36-38 ATP/hexose) leading to underestimation of active-process substrate requirements. This hinders understanding of ecological/evolutionary trade-offs between competing active processes and assessments of crop growth gains possible through bioengineering of processes that consume ATP. Determining the plant mitochondrial ATP synthase c-ring size, the degree of any minimally required (useful) bypasses of energy-conserving reactions in the respiratory chain, and the magnitude of any 'leaks' in the inner mitochondrial membrane are key research needs.

ATP modulates the activity of the voltage-gated proton channel through direct binding interaction.

ATP is an important molecule implicated in diverse biochemical processes, including the modulation of ion channel and transporter activity. The voltage-gated proton channel (Hv1) controls proton flow through the transmembrane pathway in response to membrane potential, and various molecules regulate its activity. Although it is believed that ATP is not essential for Hv1 activity, a report has indicated that cytosolic ATP may modulate Hv1. However, the detailed molecular mechanism underlying the effect of ATP on Hv1 is unknown, and whether ATP is involved in the physiological regulation of Hv1 activity remains unclear. Here, we report that cytosolic ATP is required to maintain Hv1 activity. To gain insight into the underlying mechanism, we analysed the effects of ATP on the mouse Hv1 channel (mHv1) using electrophysiological and microscale thermophoresis (MST) methods. Intracellular ATP accelerated the activation kinetics of mHv1, thereby increasing the amplitude of the proton current within the physiological concentration range. The increase in proton current was reproduced with a non-hydrolysable ATP analogue, indicating that ATP directly influences Hv1 activity without an enzymatic reaction. The direct molecular interaction between the purified mHv1 protein and ATP was analysed and demonstrated through MST. In addition, ATP facilitation was observed for the endogenous proton current flowing through Hv1 in the physiological concentration range of ATP. These results suggest that ATP influences Hv1 activity via direct molecular interactions and is required for the physiological function of Hv1. KEY POINTS: We found that ATP is required to maintain the activity of voltage-gated proton channels (Hv1) and investigated the underlying molecular mechanism. Application of intracellular ATP increased the amplitude of the proton current flowing through Hv1, accompanied by an acceleration of activation kinetics. The direct interaction between purified Hv1 protein and ATP was quantitatively analysed using microscale thermophoresis. ATP enhanced endogenous proton currents in breast cancer cell lines. These results suggest that ATP influences Hv1 activity via direct molecular interactions and that its functional characteristics are required for the physiological activity of Hv1.