Control Mechanisms for Matching ATP Supply and Demand in Heart Mitochondria

不能提供满足身体需求的能量限制了功能储备能力，在某些应激时期，如缺血，可能会导致不可逆转的细胞和组织损伤。这种匹配在心脏等代谢率高且波动迅速的组织中至关重要。线粒体是满足细胞需求的主要ATP供给者。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，其氧化还原势能反过来被电子传输链利用。电子的流量反映在氧气消耗上。这种电子流释放的能量被用来将质子从基质中输送到线粒体内膜，形成一个梯度，其质子动力驱动ATP合成酶合成ATP。这种“上游”监管被称为“推动”机制。对三磷酸腺苷合成酶的调控机制仍缺乏完整的描述。 我们发现，哺乳动物的ATP合成酶，以前被认为是一个专门运行H+的机器，实际上每一个H+利用几乎3K+来在完整的细胞线粒体内制造ATP。这种K+进入与ATP合成成正比(大约每个ATP有2 K+)，并调节基质体积，进而起到引导细胞能量利用与其产生相匹配的功能。因此，ATP合成酶首次被确定为线粒体主要的K+单一转运体，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产生的匹配的功能。 我们观察到ATP合成酶的K+电导是真实的(而不是未知污染物的人工制品)，因为在蛋白脂质体中重组的纯化F1Fo在K+梯度存在的情况下表现出稳定的(非零)膜电位，而这反过来又可以被特定的Fo抑制剂或原载体FCCP抵消。“单分子生物能量学”实验也提供了额外的证据：通过同时极微弱的光子通量检测新制造的ATP的荧光素酶生物发光和电压钳的单一K+电流，证实了在微管顶端的脂双层中重组的单个F1Fo分子纯粹由K+驱动的ATP合成，两者都被ATP合成酶的特定抑制剂阻断。使用我们为此发明的一种新技术，本实验提供了明确和明确的证据，证明在与生理K+离子环境匹配的条件下，哺乳动物ATP合成酶的单分子由K+驱动产生ATP。 为了直接和定量地评估基于蛋白脂质体重组的ATP合成酶的这些预测，并将这些观察扩展到细胞器水平，我们研究了恒定(生理)渗透压下无K+或有K+存在的大鼠心肌线粒体的生物能量表现(呼吸作用和P/O比)。利用放射性示踪剂，我们测量了在无K+或有K+的情况下，呼吸状态4和3下的体积和质子动能(PMF)的单个分量、线粒体膜电位和增量pH。总而言之，数据表明，与没有这种阳离子的情况相比，在K+存在的情况下，线粒体合成的ATP量增加了3倍(速度快1.6倍，K+/H+化学计量比几乎为3)。这些结果与用蛋白脂质体或脂双层中重组的纯化的三磷酸腺苷合成酶进行的实验所产生的预测完全一致。 我们首次证明了ATP合成酶的化学机械效率可以被上调，而且这是由某些Bcl-2家族成员和某些K+通道开放剂通过内在的ATP合成酶调节因子IF1来作用的，我们发现IF1本身是一个新的和以前未被认识的Bcl-2蛋白家族的成员。 因此，我们发现，ATP合成酶也是一种可招募的线粒体依赖的K+通道，它在细胞保护信号中发挥着关键作用，可以限制缺血再灌注损伤的损伤。因此，我们发现了两个线粒体钾通道的分子同一性，这是ATP合成酶的一个全新的功能集，也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IF1是一个新的、高度保守的BH3-2蛋白家族成员，除了BH3线性序列基序外，还显示了一个功能类似BH3结构域的分子识别特征(Morf)，使ATP合成酶功能得以调节。系统进化树表明，IF1的线性基序与BH3-Only蛋白(如Bak、Bid等)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动态平衡调节的理解。因为我们现在知道线粒体K+-单一转运体的身份是ATP合成酶，并且考虑到它主要通过K+对H+的渗透使其每天相当于人体的重量在ATP中，所以线粒体K+通量循环的实际速度和体积是巨大的(而不是以前认为的涓流泄漏)。

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).