Control Mechanisms for Matching ATP Supply and Demand in Heart Mitochondria

心脏线粒体中 ATP 供需匹配的控制机制

• 批准号: 10688767
• 负责人: Steven Sollott
• 金额: $ 287.39万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10688767
• 关键词: ATP Synthesis Pathway; Aging; Automobile Driving; BCL-2 Protein; BCL2 gene; BH3 Domain; Bioenergetics; Bioluminescence; Body Weight; Cations; Cells; Cytoprotection; Data; Detection; Development; Elderly; Electron Transport; Electrons; Energy Supply; Enzymes; Exhibits; Failure; Family; Feedback; Heart; Heart Mitochondria; Heart failure; Homeostasis; Individual; Inner mitochondrial membrane; Ions; Ischemia; Lead; Lipid Bilayers; Luciferases; Measures; Mechanics; Membrane Potentials; Mitochondria; Mitochondrial Matrix; Molecular; Morphologic artifacts; Organelles; Osmolalities; Oxidation-Reduction; Oxygen Consumption; Pathology; Performance; Persons; Photons; Phylogenetic Analysis; Physiological; Population; Potassium Channel; Potential Energy; Production; Protein Family; Proteins; Proton-Motive Force; Protons; Pump; Radioactive Tracers; Rattus; Regulation; Reperfusion Injury; Respiration; Respiratory Chain; Route; Running; Signal Transduction; Source; Specificity; Stress; Syncope; Techniques; Time; Tissues; Trees; Water; Water Movements; Workload; base; effective therapy; experimental study; feature detection; inhibitor; member; metabolic rate; mitochondrial membrane; molecular recognition; novel; pH gradient; proteoliposomes; reconstitution; recruit; restoration; single molecule; stoichiometry; voltage clamp; water channel

Failure to supply energy to match the body's demands limits the functional reserve capacity, and under certain periods of stress, such as ischemia, can lead to irreversible cell and tissue damage. This matching is critical in tissues with high and rapidly fluctuating metabolic rates such as the heart. Mitochondria are the main ATP suppliers to meet cellular demands. The fuel used by mitochondria is transported across the inner mitochondrial membrane to the matrix and produces a source of electrons whose redox-potential energy is, in turn, harnessed by the electron transport chain. The flux of electrons is reflected in oxygen consumption. The energy released from this electron flow is used to transport protons out of the matrix across the inner mitochondrial membrane forming a gradient whose proton-motive force drives ATP synthase to make ATP. This "upstream" regulation is known as the "push" mechanism. A complete description of the ATP synthase control mechanisms is still lacking. We discovered that mammalian ATP synthase, previously believed to be a machine running exclusively on H+, actually utilizes almost 3 K+ for every H+ to make ATP inside intact cellular mitochondria. This K+ entry is directly proportional to ATP synthesis (approximately 2 K+ per ATP) and regulates matrix volume, and in turn serves the function of directing the matching of cellular energy utilization with its production. Thus, ATP synthase is, for the first time, identified as a primary mitochondrial K+ uniporter, i.e., the primary way for K+ to enter mitochondria; furthermore, since this K+ entry is directly proportional to ATP synthesis and regulates matrix volume, this in turn serves the function of directing the matching of cellular energy utilization with its production. We observed that the K+ conductance of ATP synthase is real (and not an artifact of an unknown contaminant) because purified F1Fo reconstituted in proteoliposomes exhibit a stable (non-zero) membrane potential in the presence of a K+ gradient that in turn can be nulled by specific Fo inhibitors or by the protonophore FCCP. Additional proof was also provided by "single molecule bioenergetics" experiments: purely K+-driven ATP synthesis from single F1Fo molecules reconstituted in a lipid bilayer at the tip of a micropipette was demonstrated by simultaneous extreme faint-photon-flux detection of luciferase bioluminescence from newly-made ATP, and unitary K+ currents by voltage clamp, both blocked by specific inhibitors of ATP synthase. Using a novel technique that we invented for this purpose, this experiment provides unambiguous and definitive proof of K+-driven ATP production by single molecules of mammalian ATP synthase under conditions matching the physiological K+ ionic milieu. To assess directly and quantitatively these predictions based on proteoliposome reconstituted ATP synthase, and to extend these observations to the organelle level, we investigated the bioenergetic performance (respiration and P/O ratio) in the absence or presence of K+ in isolated rat heart mitochondria at constant (physiological) osmolality. Employing radioactive tracers, we measured volume, and the individual components of the protonmotive force (PMF), mitochondrial membrane potential and delta-pH, in the absence or presence of K+ under states 4 and 3 respiration. Together, the data indicate that mitochondria synthesize 3-fold higher amounts of ATP (at 1.6-fold faster rates, and a K+/H+ stoichiometry of almost 3) in the presence of K+ as compared to conditions in which this cation is absent. These results are fully consistent with predictions arising from experiments performed with purified ATP synthase reconstituted in proteoliposomes or lipid bilayers. For the first time, we show that the chemo-mechanical efficiency of ATP synthase can be up-regulated, and that this occurs by certain members of the Bcl-2 family and by certain K+ channel openers acting via an intrinsic regulatory factor of ATP synthase, IF1, which we identified as itself a novel and previously unrecognized member of the Bcl-2 protein family. As a consequence of the foregoing, we discovered that ATP synthase is also a recruitable mitochondrial ATP-dependent K+ channel which serves critical functions in cell protection signaling that can limit the damage of ischemia-reperfusion injury. Thus, we discovered the molecular identity of two mitochondrial potassium channels, an entirely new function set for ATP synthase, and what is likely the primary mechanism by which mitochondrial function matches energy supply with demand for all cells in the body. We discovered that IF1 is a novel, highly conserved BH3-only member of the Bcl-2 protein family displaying, in addition to the BH3 linear sequence motif, a functional BH3-domain-like molecular recognition feature (MoRF) which enables the modulation of ATP synthase function. The phylogenetic tree shows that IF1s linear motif is most closely related to the BH3-only proteins (e.g. Bak, Bid, etc.). These findings will fundamentally change our understanding of the regulation of mitochondrial energy production and homeostasis. Because we now know the identity of the mitochondrial K+-uniporter to be the ATP synthase, and given its dominant permeation by K+ over H+ to make the daily equivalent of the body's weight in ATP, the actual rate and volume of mitochondrial K+ flux cycling is huge (and not the previously believed trickle-leak). Although there is a much smaller abundance of cytoplasmic Na+ (in comparison to K+) we discovered that ATP synthase also utilizes Na+ to make ATP (and hence transports it in proportion to ATP synthesis), which in turn engages a novel mechanism we have discovered to regulate mitochondrial Ca2+ retention and in turn, augmenting Ca2+-activated enzymes driving mitochondrial energy production. We have also been working to characterize whether and how these novel ATP synthase mechanisms change with aging. K+ influx through ATP synthase is directly proportional to ATP synthesis so that changes in ATP utilization (workload) are accompanied by transient increases of intramitochondrial K+ which, in turn, produces osmotic changes that positively regulates matrix volume and respiratory chain activity. This positively regulated respiratory chain activity facilitates restoration of the just-utilized membrane potential and delta-pH (the energy needed to drive ATP synthesis), and is necessary to match ATP energy production to its demand. Although mitochondria are known to have aquaporin water channels that serve to passively move water driven by osmotic changes, we have found high rates and magnitudes of the actual dynamic mitochondrial volume changes during transitions between low to high workloads that may be beyond the capacity of passive water movement at the (modest) expression levels of mitochondrial aquaporins. Therefore, the known routes of water transport in mitochondria appear to be insufficient to explain the dynamism of physiologic energy supply-demand transitions, and we are examining novel routes of mitochondrial water transport to explain the actual matrix volume dynamics. We discovered that individual water molecules may be co-transported through F1Fo with each H+ and K+ ion leading to the dynamic flux of water into the mitochondrial matrix in direct proportion to ATP production. This proves the novel mechanism that mitochondrial F1Fo may serve as an active water pump, and the water moved would serve as a regulatory matrix-volume feedback signal to activate respiration, facilitating energy supply-demand matching. Thus, the ability of mitochondrial ATP synthesis to meet rapid changes in energy demand depends on active water transport through ATP synthase.