Biophysics of Large Membrane Channels

大膜通道的生物物理学

• 批准号: 6991143
• 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/6991143
• 关键词: Bax gene /protein; apoptosis; biophysics; cytochrome c; ion transport; ionic bond; lipid bilayer membrane; membrane channels; mitochondria; polymers; protein purification

We investigate the physical principles of channel-facilitated transport of metabolites and other large solutes across cell and organelle membranes. To study channels under precisely controlled conditions, we reconstitute channel-forming proteins into planar lipid bilayers. Using this strategy, we elucidate the molecular mechanisms responsible for metabolite flux regulation under normal conditions and in pathology. I. Apoptosis: VDAC Regulation by Bcl-2 Family of Proteins. Mitochondria?s crucial role in the initiation of apoptosis is well established. Triggered by a number of different stimuli, the mitochondrial outer membrane (MOM) becomes permeable to apoptogenic factors such as cytochrome c. The Bcl-2 family proteins regulate the permeabilization of MOM: pro-apoptotic proteins such as Bax and Bid induce the release of apoptogenic factors, whereas anti-apoptotic proteins such as Bcl-xL prevent their release. There are two working models that associate these proteins with the existing major channel in the MOM, VDAC. Responsible for most of the metabolite flux across MOM, this channel is a very attractive candidate for a pathway for cytochrome c release. In the first model, the pro-apoptotic protein, Bax, induces formation of the large VDAC-based channels permeable to cytochrome c. In the second model it is the closure of VDAC channels that leads to MOM rupture. Therefore, though in both models the ability of Bcl-2 proteins to regulate the state and integrity of VDAC explains their function as either anti- or pro-apoptotic agents, the proposed mechanisms of VDAC channel regulation are diametrically opposite. To address this question we studied the effect of these proteins on the properties of VDAC channels reconstituted into the planar phospholipid membranes. First, we have demonstrated that, contrarily to general belief, there is no functionally significant interaction between VDAC channels and pro-apoptotic protein Bax in either monomeric or oligomeric forms. A detailed analysis of the characteristic properties of VDAC channels such as voltage gating, ion selectivity, single channel conductance, and water-soluble polymer exclusion did not show any change after Bax addition regardless of lipid composition, medium pH, or ionic content. However, we have found that another pro-apoptotic protein, tBid, affects the voltage-gating properties of VDAC by inducing channel closure. We have shown here that tBid induces closure of VDAC channels, both on single and multichannel membranes, in a dose-dependent manner. By decreasing the probability of VDAC opening, tBid would reduce the flux of adenine nucleotides and other negatively charged metabolites across the MOM, which may affect mitochondrial functions in different ways. One of the possible consequences of VDAC channel closure is the disruption of metabolite exchange across the MOM and the resultant accumulation of metabolites in the intermembrane space followed by mitochondria swelling and consequent loss of MOM integrity. The mechanism by which tBid alters the gating properties of VDAC remains to be understood. II. Water-Soluble Polymers as Molecular Probes. Polymer partitioning into nanoscale cavities is crucially important in many areas of science and technology. During the past year we have extended our studies in this direction to address the problem of polymer solution non-ideality. Thermodynamics of polymer partitioning in the regime of advanced solution non-ideality (de Cloizeaux regime) was studied with the alpha-Hemolysin channel. Using the change in conductance of a nanometer-wide protein pore of this channel to detect pore occupancy by polymers, we measured the equilibrium partitioning of differently sized linear poly(ethylene glycol)s (PEGs) as a function of polymer concentration in the bulk solution. In the semidilute regime, increased polymer concentration resulted in a sharp increase in polymer partitioning. Quantifying solution non-ideality by osmotic pressure and taking the free energy of polymer confinement by the pore at infinite dilution as an adjustable parameter allowed us to describe polymer partitioning only at low polymer concentrations. At larger concentrations the increase in partitioning is much sharper than the model predictions. The nature of this sharp transition between strong exclusion and strong partitioning might be rationalized within the concepts of scaling theory predicting this kind of behavior whenever the correlation length of the monomer density in the semidilute bulk solution becomes smaller than the pore radius. Specific attractive interactions between the protein pore and the polymer that exist in addition to the entropic repulsion accounted for in the present study may also play a role. III. Electrostatics in Ion Transport through Large Channels. Although the crystallographic structure of the bacterial porin OmpF has been known for a decade, the physical mechanisms of its ionic selectivity are still under investigation. We address this issue in a series of experiments with varied pH, salt concentrations, inverted salt gradient, and charged and uncharged lipids. Measuring reversal potential, we show that OmpF selectivity (traditionally regarded as slightly cationic) depends strongly on pH and salt concentration and is conditionally asymmetric, that is, sensitive to the direction of salt concentration gradient. At neutral pH and sub-decimolar salt concentrations the channel exhibits nearly ideal cation selectivity. Substituting neutral DPhPC with DPhPS, we demonstrate that the fixed charge of the host lipid has a small but measurable effect on the channel reversal potential. The available structural information allows for a qualitative explanation of our experimental findings. These findings now lead us to re-examine the ionization state of 102 titratable sites lining the OmpF channel. Using standard methods of continuum electrostatics tailored to our particular purpose, we find the charge distribution in the channel as a function of solution acidity and relate the pH-dependent asymmetry in channel selectivity to the pH-dependent asymmetry in charge distribution. In an attempt to find a simple phenomenological description of our results, we also discuss different macroscopic models of electrodiffusion through large channels.

我们研究了代谢产物和其他大溶质跨细胞和细胞器膜的通道相关转运的物理原理。为了在精确控制的条件下研究通道，我们将形成的通道形成蛋白重新构建为平面脂质双层。使用这种策略，我们阐明了在正常条件和病理学中负责代谢物通量调节的分子机制。 I.凋亡：Bcl-2蛋白质家族的VDAC调节。线粒体在凋亡的开始中的关键作用已建立。由许多不同的刺激触发，线粒体外膜（MOM）可渗透到诸如细胞色素c之类的凋亡因子。 Bcl-2家族蛋白调节MOM的通透性：促凋亡蛋白（例如BAX和BID）诱导凋亡因子的释放，而抗凋亡蛋白（如BCL-XL）则阻止其释放。有两个工作模型将这些蛋白质与妈妈VDAC中现有的主要通道相关联。该通道负责大多数代谢物通量，是细胞色素C释放途径的非常有吸引力的候选者。在第一个模型中，促凋亡蛋白Bax诱导了可渗透到细胞色素c的大型基于VDAC的通道的形成。在第二个模型中，VDAC通道的关闭导致了MOM破裂。因此，尽管在这两种模型中，Bcl-2蛋白调节VDAC状态和完整性的能力解释了它们作为抗凋亡剂或促凋亡剂的功能，但VDAC通道调节的提议的机制截然相反。为了解决这个问题，我们研究了这些蛋白质对在平面磷脂膜中重构VDAC通道的特性的影响。首先，我们已经证明，与一般信念相反，单体或寡聚形式中VDAC通道与促凋亡蛋白BAX之间在功能上没有显着的相互作用。对VDAC通道的特性特性（例如电压门控，离子选择性，单通道电导和水溶性聚合物排除）的详细分析并未显示出Bax添加后的任何变化，而不管脂质组成，中等pH或离子含量如何。但是，我们发现另一种促凋亡蛋白TBID通过诱导通道闭合影响VDAC的电压门控性能。我们在这里已经显示，TBID以剂量依赖性方式诱导了在单个和多通道膜上的VDAC通道的闭合。通过降低VDAC开放的概率，TBID将减少腺苷核苷酸和其他带负电的代谢产物的通量，这可能会以不同的方式影响线粒体功能。 VDAC通道关闭的可能后果之一是整个妈妈的代谢产物交换的破坏以及代谢物在膜间空间中的积累，随后是线粒体肿胀以及随之而来的MOM完整性的丧失。 TBID改变VDAC的门控性能的机制仍有待理解。 ii。水溶性聚合物作为分子探针。在科学技术的许多领域，将聚合物分配到纳米级腔中至关重要。在过去的一年中，我们将研究扩展到了这个方向，以解决聚合物溶液的非理想性问题。用α-蛋白酶通道研究了晚期溶液非理想性（DE Cloizeaux制度）中聚合物分配的热力学。利用该通道的纳米范围蛋白孔的电导变化来检测聚合物的孔占用率，我们测量了不同尺寸的线性聚（乙二醇）S（PEG）S（PEGS）S（PEGS）S的平衡分配，作为大量溶液中聚合物浓度的函数。在半硅酸酯方面，聚合物浓度的增加导致聚合物分配急剧增加。通过渗透压量化溶液的非理想性，并在无限稀释下将孔限制为可调参数的聚合物限制，这使我们能够仅在低聚合物浓度下描述聚合物分配。在较大的浓度下，分配的增加比模型预测更明显。在缩放理论的概念概念中，这种急剧排除和强分区之间的急剧过渡的性质可能会被合理化，每当半硅散装溶液中单体密度的相关长度变得小于孔径时，就可以预测这种行为。蛋白质孔和聚合物之间存在的特定有吸引力的相互作用除了在本研究中造成的熵排斥外，也可能起作用。 iii。离子通过大通道传输的静电。尽管细菌孔蛋白OMPF的晶体学结构已知十年，但其离子选择性的物理机制仍在研究中。我们在一系列具有不同pH，盐浓度，倒盐梯度以及带电和无电的脂质的实验中解决了这个问题。测量逆转电位，我们表明OMPF选择性（传统上被视为阳性略有阳离子）在很大程度上取决于pH和盐浓度，并且有条件地不对称，即对盐浓度梯度的方向敏感。在中性pH值和亚透膜盐浓度下，通道表现出几乎理想的阳离子选择性。用DPHP将中性DPHPC取代，我们证明了宿主脂质的固定电荷对通道反转电位的影响很小但可测量。可用的结构信息允许对我们的实验发现进行定性解释。这些发现现在使我们重新检查了OMPF通道内置的102个可滴定位点的电离状态。使用针对我们特定目的量身定制的连续静电静电的标准方法，我们发现通道中的电荷分布是溶液酸度的函数，并将通道选择性中的pH依赖性不对称性与电荷分布中的pH依赖性不对称性相关联。为了找到对我们结果的简单现象学描述，我们还通过大通道讨论了电辐射的不同宏观模型。