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: 10005755
• 负责人: Steven Sollott
• 金额: $ 87.4万
• 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/10005755
• 关键词: ATP Synthesis Pathway; BCL-2 Protein; BCL2 gene; BH3 Domain; Bioenergetics; Bioluminescence; Body Weight; Cations; Cells; Cytoprotection; Data; Detection; Development; Elderly; Electron Transport; Electrons; Energy Supply; Exhibits; Failure; Family; Heart; Heart Mitochondria; Heart failure; Homeostasis; Individual; Inner mitochondrial membrane; Ischemia; Lead; Lipid Bilayers; Luciferases; Measures; Mechanics; Membrane Potentials; Mitochondria; 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; Radioactive Tracers; Rattus; Regulation; Reperfusion Injury; Respiration; Running; Signal Transduction; Source; Specificity; Stress; Syncope; Techniques; Time; Tissues; Trees; base; effective therapy; experimental study; feature detection; inhibitor/antagonist; member; metabolic rate; mitochondrial membrane; molecular recognition; novel; pH gradient; proteoliposomes; reconstitution; recruit; single molecule; stoichiometry; voltage clamp

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).

不能提供能量以满足身体的需求限制了功能储备能力，并且在某些压力时期，如缺血，可能导致不可逆的细胞和组织损伤。这种匹配在具有高且快速波动的代谢率的组织（如心脏）中至关重要。线粒体是满足细胞需求的主要ATP供应者。线粒体使用的燃料通过线粒体内膜运输到基质，并产生电子源，电子源的氧化还原势能反过来又被电子运输链利用。电子的通量反映在氧的消耗上。从该电子流释放的能量用于将质子运输出基质穿过线粒体内膜，形成梯度，其质子动力驱动ATP合酶产生ATP。这种“上游”调控被称为“推动”机制。ATP合成酶的控制机制的完整描述仍然缺乏。 我们发现，哺乳动物的ATP合成酶，以前被认为是一个机器上运行的H+，实际上利用几乎3 K+为每个H+，使完整的细胞线粒体内的ATP。这种K+进入与ATP合成成正比（每个ATP约2 K+），并调节基质体积，进而起到指导细胞能量利用与其生产相匹配的作用。因此，ATP合酶首次被鉴定为主要的线粒体K+单向转运体，即，K+进入线粒体的主要途径;此外，由于这种K+进入与ATP合成成正比并调节基质体积，这反过来又起到指导细胞能量利用与其生产相匹配的作用。 我们观察到ATP合酶的K+电导是真实的（而不是未知污染物的伪像），因为在蛋白脂质体中重构的纯化的F1 Fo在K+梯度存在下表现出稳定的（非零）膜电位，而K+梯度又可以被特定的Fo抑制剂或质子载体FCCP抵消。 “单分子生物能学”实验也提供了额外的证据：通过同时检测新制备的ATP的荧光素酶生物发光的极弱光子通量和电压钳的单一K+电流，证明了由微量移液管尖端脂质双层中重构的单个F1 Fo分子的纯K+驱动的ATP合成，两者都被ATP合酶的特异性抑制剂阻断。使用一种新的技术，我们发明了这个目的，这个实验提供了明确的和明确的证据K+驱动的ATP生产的哺乳动物ATP合酶的单分子的条件下匹配的生理K+离子环境。 为了直接和定量地评估这些预测的基础上，蛋白脂质体重建的ATP合酶，并将这些观察扩展到细胞器的水平，我们研究了生物能量性能（呼吸和P/O比）的情况下或存在K+在离体大鼠心脏线粒体在恒定（生理）渗透压。采用放射性示踪剂，我们测量的体积，和质子动力（PMF），线粒体膜电位和Δ-pH值的各个组件，在K+的存在或不存在下的状态4和3呼吸。总之，数据表明，与不存在该阳离子的条件相比，在K+存在下，线粒体合成3倍高量的ATP（以快1.6倍的速率，并且K+/H+化学计量几乎为3）。这些结果是完全一致的预测所产生的实验与纯化的ATP合酶重组蛋白脂质体或脂质双层。 对于第一次，我们表明，ATP合酶的化学机械效率可以上调，这是由Bcl-2家族的某些成员和某些K+通道开放剂通过ATP合酶，IF 1，我们确定为本身的一种新的和以前未识别的Bcl-2蛋白家族的成员的内在调节因子的作用。 作为上述的结果，我们发现ATP合酶也是一种可募集的线粒体ATP依赖性K+通道，其在细胞保护信号传导中起关键作用，所述细胞保护信号传导可以限制缺血-再灌注损伤的损害。因此，我们发现了两个线粒体钾通道的分子特性，这是ATP合酶的一个全新功能集，并且可能是线粒体功能将能量供应与体内所有细胞的需求相匹配的主要机制。 我们发现，IF 1是一种新的，高度保守的BH 3-唯一的Bcl-2蛋白家族的成员展示，除了BH 3线性序列基序，功能BH 3结构域样分子识别功能（MoRF），使ATP合酶功能的调制。系统进化树显示IF 1的线性基序与BH 3-only蛋白（如巴克、Bid等）的关系最为密切。 这些发现将从根本上改变我们对线粒体能量产生和稳态调节的理解。因为我们现在知道线粒体K+单向转运体是ATP合成酶，并且考虑到其主要渗透是K+超过H+，以使ATP每日相当于体重，线粒体K+通量循环的实际速率和体积是巨大的（而不是以前认为的涓滴泄漏）。