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.

无法提供满足身体需求的能量会限制功能储备能力，并且在某些应激时期（例如缺血）下可能会导致不可逆的细胞和组织损伤。这种匹配对于代谢率高且快速波动的组织（例如心脏）至关重要。线粒体是满足细胞需求的主要 ATP 供应商。线粒体使用的燃料穿过线粒体内膜输送到基质，并产生电子源，其氧化还原势能反过来被电子传输链利用。电子通量反映在耗氧量上。电子流释放的能量用于将质子从基质中穿过线粒体内膜，形成梯度，其质子动力驱动 ATP 合酶产生 ATP。这种“上游”调节被称为“推动”机制。仍然缺乏 ATP 合酶控制机制的完整描述。 我们发现，哺乳动物 ATP 合酶（之前被认为是仅在 H+ 上运行的机器）实际上利用每个 H+ 近 3 K+ 在完整的细胞线粒体内产生 ATP。该 K+ 条目与 ATP 合成成正比（每个 ATP 大约 2 K+）并调节基质体积，进而起到指导细胞能量利用与其产生相匹配的功能。因此，ATP合酶首次被确定为主要的线粒体K+单向转运蛋白，即K+进入线粒体的主要途径；此外，由于该 K+ 条目与 ATP 合成成正比并调节基质体积，因此这反过来又起到指导细胞能量利用与其产生相匹配的功能。 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影），因为在蛋白脂质体中重构的纯化 F1Fo 在存在 K+ 梯度的情况下表现出稳定的（非零）膜电位，而 K+ 梯度又可以为零通过特定的 Fo 抑制剂或通过质子载体 FCCP。 “单分子生物能学”实验还提供了额外的证据：通过对新的荧光素酶生物发光进行同时极端微弱光子通量检测，证明了在微量移液器尖端的脂质双层中重构的单个 F1Fo 分子的纯 K+ 驱动的 ATP 合成。产生的 ATP 和通过电压钳产生的单一 K+ 电流，均被 ATP 合酶的特定抑制剂阻断。使用我们为此目的发明的新技术，该实验提供了哺乳动物 ATP 合酶单分子在与生理 K+ 离子环境相匹配的条件下 K+ 驱动的 ATP 产生的明确且明确的证据。 为了直接定量地评估这些基于蛋白脂质体重构 ATP 合酶的预测，并将这些观察结果扩展到细胞器水平，我们研究了分离的大鼠心脏线粒体中不存在或存在 K+ 时的生物能性能（呼吸和 P/O 比）。恒定（生理）渗透压。使用放射性示踪剂，我们测量了在状态 4 和状态 3 呼吸下存在或不存在 K+ 的情况下的体积以及质子动力 (PMF) 的各个组成部分、线粒体膜电位和 δ-pH。总之，数据表明，与不存在 K+ 的条件相比，在 K+ 存在的情况下，线粒体合成的 ATP 量高出 3 倍（速度快 1.6 倍，K+/H+ 化学计量几乎为 3）。这些结果与用蛋白脂质体或脂质双层中重构的纯化 ATP 合酶进行的实验所得出的预测完全一致。 我们首次证明 ATP 合酶的化学机械效率可以被上调，并且这是通过 Bcl-2 家族的某些成员和某些 K+ 通道开放剂通过 ATP 合酶的内在调节因子起作用而发生的, IF1，我们将其本身确定为 Bcl-2 蛋白家族的一个新的且以前未被识别的成员。 由于上述原因，我们发现 ATP 合酶也是一种可招募的线粒体 ATP 依赖性 K+ 通道，其在细胞保护信号传导中发挥关键功能，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、ATP 合酶的全新功能集，以及线粒体功能将体内所有细胞的能量供应与需求相匹配的主要机制。 我们发现 IF1 是 Bcl-2 蛋白家族中一种新型、高度保守的仅 BH3 成员，除了 BH3 线性序列基序外，还显示功能性 BH3 结构域样分子识别特征 (MoRF)，该特征能够调节ATP合酶功能。系统发育树显示 IF1 线性基序与 BH3-only 蛋白（例如 Bak、Bid 等）关系最密切。 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+ 单向转运蛋白是 ATP 合酶，并且考虑到它主要通过 K+ 而非 H+ 渗透，以产生相当于人体体重的每日 ATP，线粒体 K+ 通量循环的实际速率和体积为巨大的（而不是以前认为的滴漏）。 尽管细胞质 Na+ 的丰度要小得多（与 K+ 相比），但我们发现 ATP 合成酶也利用 Na+ 来制造 ATP（因此按照 ATP 合成的比例输送它），这反过来又涉及我们发现的一种新机制调节线粒体 Ca2+ 保留，进而增强 Ca2+ 激活酶驱动线粒体能量产生。我们还一直致力于研究这些新型 ATP 合酶机制是否以及如何随着衰老而变化。 通过 ATP 合成酶的 K+ 流入与 ATP 合成成正比，因此 ATP 利用率（工作量）的变化伴随着线粒体内 K+ 的短暂增加，这反过来又产生渗透压变化，从而积极调节基质体积和呼吸链活性。这种正向调节的呼吸链活动有助于恢复刚刚利用的膜电位和 δ-pH（驱动 ATP 合成所需的能量），并且对于使 ATP 能量产生与其需求相匹配是必要的。尽管已知线粒体具有水通道蛋白水通道，可在渗透压变化驱动下被动移动水，但我们发现在低工作负荷到高工作负荷之间的过渡期间，实际动态线粒体体积变化的速率和幅度很高，这可能超出了被动水的能力线粒体水通道蛋白（适度）表达水平的运动。因此，线粒体中已知的水运输途径似乎不足以解释生理能量供需转变的动态，我们正在研究线粒体水运输的新途径以解释实际的基质体积动力学。 我们发现单个水分子可能通过 F1Fo 与每个 H+ 和 K+ 离子共同转运，导致水动态流入线粒体基质，与 ATP 产量成正比。这证明了线粒体F1Fo可能作为主动水泵的新机制，移动的水将作为调节基质体积反馈信号来激活呼吸，促进能量供需匹配。因此，线粒体 ATP 合成满足能量需求快速变化的能力取决于通过 ATP 合酶的活跃水运输。