Biophysics of Large Membrane Channels

大膜通道的生物物理学

• 批准号: 7198243
• 负责人: sergey bezrukov
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 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/7198243
• 关键词: Bax gene /protein; apoptosis; biophysics; cytochrome c; ion transport; ionic bond; lipid bilayer membrane; membrane channels; membrane transport proteins; mitochondrial membrane; polymers; protein purification; protein reconstitution; voltage gated channel

Large ion channels are key structural elements of metabolite exchange between different cellular compartments and between cells. To study these channels under precisely controlled conditions, we reconstitute channel-forming proteins into planar lipid bilayers. Our goal is to investigate the physical principles of channel-facilitated transport of metabolites and other large solutes across cell and organelle membranes. I. Apoptosis. Research on VDAC, the major channel from mitochondria outer membrane (MOM), has accelerated as evidence grows of its importance in mitochondrial function and in apoptosis. This small, ancient, highly-conserved protein appears to be involved in many cellular processes. The past year?s effort was to shed light on its role in apoptosis and to separate reliable information from more questionable claims. VDAC channels can exist in a variety of functional states that differ in their ability to pass non-electrolytes and conduct ions. The permeability of VDAC to small ions includes Ca. It is well established fact that cytosolic Ca triggers opening of the permeability transition pore (PTP) in the mitochondria inner membrane, which consequently allows passage of water and solutes up to approximately 1.5 kDa. Opening of PTP is one of the mechanisms responsible for MOM permeabilization, cytochrome c release, and, consequently, the apoptotic cell death. VDAC is thought to be one of the major components of the PTP. If VDAC is part of PTP, then it seems logical that closure of VDAC would close the PTP and protect mitochondria from cytosolic PTP activators, such as Ca ions. In experiments with VDAC channel reconstituted unto the planar lipid membranes we have shown that Ca permeates through both the open and ?closed? states of VDAC. The double positive charge does not exclude Ca cations from the open state because the anion selectivity is moderate. The closed state favors cations. The presence or absence of Ca does not change the conductance of the open state of VDAC. In 1 M NaCl the conductance is 3.3 +/- 0.3 nS in the presence of 0.1 mM EGTA and 3.4 +/- 0.1 nS in the presence of 1 mM CaCl2. Moreover, we have also shown that the voltage gating is unaffected by Ca presence. Closure of VDAC does not prevent Ca flux and the closed state of VDAC has a similar molecular size cut-off as PTP. Our conclusion is that the closure of VDAC cannot protect against PTP opening. Thus, Ca cannot regulate PTP by opening or closing VDAC. The notion of a supramolecular PTP complex is fashionable but seems unnecessary considering the large number of VDAC channels in the outer membrane and the higher permeability of VDAC than that measured for the PTP. Correspondingly, the influx of metabolites that leads to the swelling of the matrix must flow through VDAC whether the supramolecular complex exists or not. These data and the analysis of the results obtained by other groups confirm our previously suggested model wherein closure of VDAC, not VDAC opening, leads to MOM permeabilization and apoptosis. II. Water-Soluble Polymers as Molecular Probes. The past year?s progress in quantitative understanding of polymer probing of ion channels resulted in a theoretical model explaining the observed polymer concentration dependence of the partition coefficient. We achieved this by recognizing that non-ideality of polymer solution in the pore is weaker than in the bulk because the overlap volume fraction of the polymer in the pore is higher than that in the bulk. The reason is that polymer molecules in the pore form cigars with high intra-molecular monomer density. Therefore, the observed concentration dependence of the partition coefficient cannot be explained using the standard approach that assumes non-ideality of the polymer solution in the pore to be identical to non-ideality in the bathing solution: measured partitioning is a much sharper function of polymer concentration than identical non-ideality predicts. In a separate study, we also analyzed the data on the electrical conductivity and viscosity of aqueous solutions of polyethylene glycol in order to use them as the reference information in studying channels. Over wide ranges of concentration and polymer molecular weight, conductivity is independent of the molecular weight for long chains and weakly dependent on the molecular weight for short chains. The processes responsible for a decrease in the mobility of ions were qualitatively analyzed to explain the weak conductivity sensitivity to the length of polymer chains. It was shown that experiments can be interpreted using the microviscosity concept. Microviscosity increases with the addition of a polymer much less rapidly than usual (macroscopic) viscosity. A simple empirical formula describing the dependence of conductivity on the polymer concentration was suggested. III. Transport Properties of Toxin Channels. Understanding of the physical principles and structural aspects involved in large-channel permeability and selectivity is still far from being satisfactory, and our progress relies on the detailed knowledge of the transport phenomenology. This knowledge can be obtained in experiments with single channels reconstituted into planar bilayers by studying the effect of penetrating molecules on the ionic current through the channel. High-resolution conductance recording and appropriate statistical analyses allow one to quantitatively evaluate solute partitioning into, and dynamics within, the confines of ion channel aqueous pores. During the past year we studied how molecular topology of polyethylene glycol (PEG) influences its interaction with large channels. We found that closing linear PEG into a circular ?crown? molecule dramatically changes its dynamics in the alpha-Hemolysin channel. In the electrically neutral crown ether, six ethylene oxide monomers are linked into a circle that gives the molecule ion-complexing capacity and increases its rigidity. As with linear PEG, addition of the crown to the membrane-bathing solution decreases the ionic conductance of the channel and generates additional conductance noise. However, in contrast to linear PEG, both the conductance reduction (reporting on crown partitioning into the channel pore) and the noise (reporting on crown dynamics in the pore) now depend strongly and non-monotonically on the applied voltage. Within the whole frequency range accessible by channel reconstitution experiments, the noise power spectrum is ?white?, showing that crown exchange between the channel and the bulk solution is fast. Analyzing these data in the framework of a Markovian two-state model, we are able to characterize the process quantitatively. We show that the lifetime of the crown in the channel pore reaches its maximum (a few microseconds) at about the same voltage (approximately 100 mV, negative from the side of protein addition) where the crown?s reduction of the channel conductance is most pronounced. Our interpretation is that, because of its rigidity, the crown feels an effective steric barrier in the narrowest part of the channel pore. This barrier together with crown-ion complexing and resultant interaction with the applied field leads to behavior usually associated with voltage-dependent binding in the channel pore. Studies of crown ether effects on the conductance of ion channels are important because the crown ether superfamily has been shown to exhibit pharmacological effects.

大离子通道是不同细胞室和细胞之间代谢物交换的关键结构元素。为了在精确控制的条件下研究这些通道，我们将形成通道的蛋白质重新构建为平面脂质双层。我们的目标是研究代谢产物和其他大质溶质在细胞和细胞器膜中的渠道转运运输的物理原理。 I.凋亡。线粒体外膜（MOM）的主要通道VDAC的研究已加速，因为证据增长了其在线粒体功能和凋亡中的重要性。这种古老的，高度保存的蛋白质似乎参与了许多细胞过程。过去一年的努力是阐明其在凋亡中的作用，并将可靠的信息与更可疑的主张分开。 VDAC通道可以存在于多种功能状态，它们通过非电解质和导电离子的能力有所不同。 VDAC对小离子的渗透性包括CA。良好确定的事实是，在线粒体内膜中，胞质CA触发了渗透率过渡孔（PTP）的打开，从而允许水并溶于大约1.5 kDa。 PTP的开放是负责MOM通透性，细胞色素C释放以及凋亡细胞死亡的机制之一。 VDAC被认为是PTP的主要组成部分之一。如果VDAC是PTP的一部分，那么VDAC的闭合将关闭PTP并保护线粒体免受胞质PTP激活剂（例如CA离子）的影响似乎是合乎逻辑的。在使用VDAC通道进行的实验中，我们已经向平面脂质膜进行了重构，我们表明CA渗透到开放式和闭合？ VDAC州。由于阴离子的选择性适中，双重电荷不会从开放状态排除CA阳离子。封闭的国家有利于阳离子。 CA的存在或不存在不会改变VDAC开放状态的电导。在1 m NaCl中，在存在1 mM CaCl2的情况下，在0.1 mM EGTA和3.4 +/- 0.1 ns的情况下，电导为3.3 +/- 0.3 ns。此外，我们还表明，电压门口不受CA存在影响。 VDAC的闭合不会防止CA通量，并且闭合VDAC的态度的分子大小与PTP相似。我们的结论是，VDAC的关闭无法防止PTP打开。因此，CA不能通过打开或关闭VDAC来调节PTP。超分子PTP复合物的概念是时尚的，但考虑到外膜中的VDAC通道数量大，而VDAC的渗透性大于PTP测得的VDAC。相应地，无论是否存在是否存在超分子复合物，导致基质肿胀的代谢产物的流入都必须流过VDAC。这些数据以及对其他组获得的结果的分析证实了我们先前建议的模型，其中VDAC的闭合（而不是VDAC）会导致MOM通透性和凋亡。 ii。水溶性聚合物作为分子探针。过去一年对离子通道聚合物探测的定量理解的进展，导致了一个理论模型，解释了观察到的分区系数的聚合物浓度依赖性。我们通过认识到孔中聚合物溶液的非理想性比在大体中弱，因为孔中聚合物的重叠体积分数高于大块中的重叠体积分数。原因是孔中的聚合物分子形成雪茄，具有高分子内单体密度。因此，无法使用标准方法来解释观察到的分区系数的浓度依赖性，该方法假设孔中聚合物溶液的非理想性与沐浴溶液中的非理想性相同：测得的分配是聚合物浓度比相同的非思想性预测的要敏锐的。在另一项研究中，我们还分析了聚乙烯乙二醇水溶液的电导率和粘度的数据，以便将它们用作研究通道中的参考信息。在浓度和聚合物分子量的范围内，电导率与长链的分子量无关，而弱依赖于短链的分子量。定性分析了导致离子迁移率降低的过程，以解释电导率对聚合物链长度的较弱敏感性。结果表明，可以使用微粘度概念来解释实验。与平常（宏观）粘度的添加速度差得多，微度粘度会增加。提出了一个简单的经验公式，描述了电导率对聚合物浓度的依赖性。 iii。毒素通道的转运特性。了解大通道通透性和选择性所涉及的物理原理和结构方面仍然远非令人满意，我们的进步依赖于运输现象学的详细知识。可以通过研究穿透分子对通过通道的离子电流的影响，在与单个通道重构为平面双层的实验中获得这些知识。高分辨率电导记录和适当的统计分析允许人们定量评估离子通道水孔内部范围内的溶质分配和动力学。在过去的一年中，我们研究了聚乙烯乙二醇（PEG）的分子拓扑如何影响其与大通道的相互作用。我们发现闭合线性钉成圆形？分子在Alpha-Hemolysin通道中急剧改变其动力学。在电气中性的冠状醚中，将六个乙烷氧化物单体连接到一个圆圈中，从而使分子离子 - 复合能力并增加其刚度。与线性PEG一样，将冠添加到膜式式溶液中会降低通道的离子电导率并产生额外的电导噪声。然而，与线性钉相比，现在的电导率降低（有关冠状孔的报告）和噪声（关于孔中冠状动力学的噪声）现在非常依赖于施加的电压。在整个频率范围内，可以通过通道重建实验访问，噪声功率谱是白色？，显示通道和批量解决方案之间的冠状交换很快。在马尔可夫两国模型的框架中分析这些数据，我们能够定量地表征该过程。我们表明，通道孔中冠的寿命达到其最大值（几微秒）的最大电压（约100 mV，含蛋白质添加的侧面为100 mV），其中冠状电通道电导率的降低最为明显。我们的解释是，由于其刚性，皇冠在通道孔的最狭窄部分中感觉到有效的空间屏障。该障碍与牙冠离子络合物以及与所施加的场相互作用的相互作用导致通常与通道孔中电压依赖性结合有关的行为。对离子通道电导的影响的研究很重要，因为已显示冠状超家族表现出药理学作用。