Biophysics of Large Membrane Channels

大膜通道的生物物理学

基本信息

批准号：
8553824
负责人：
sergey bezrukov
金额：
$ 103.38万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
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/8553824
关键词：
Address Alamethicin Anesthetics Bacillus anthracis Bacterial Toxins Behavior Binding Biophysics Caliber Cell Proliferation Cell membrane Cells Characteristics Charge Chemistry Cholesterol Clinical Clostridium perfringens Clostridium perfringens epsilon toxin Colchicine Complex Confocal Microscopy Cyclic AMP-Dependent Protein Kinases Cytoskeletal Proteins Cytoskeleton Cytosol Data Dependence Development Diffuse Disease Drug Delivery Systems Entropy Equilibrium Escherichia coli Glycogen Synthase Kinases Glycolysis Goals Halothane Health Hemolysin Human In Vitro Inhalation Anesthetics Integral Membrane Protein Ion Channel Isotopes Learning Lipid Bilayers Lipids Measures Mediating Membrane Membrane Lipids Membrane Microdomains Membrane Proteins Microbial Biofilms Microtubule Polymerization Microtubules Mitochondria Modeling Molecular Motion Neutron Diffraction New Agents Oils Operative Surgical Procedures Organelles Organism Outer Mitochondrial Membrane Oxidative Phosphorylation Peptides Permeability Pharmaceutical Preparations Phase Phosphorylation Physics Physiological Physiology Play Property Proteins Pseudomonas aeruginosa Pseudomonas syringae Recording of previous events Regulation Relative (related person)Research Research Personnel Residual state Roentgen Rays Scientist Shapes Signal Pathway Signal Transduction Solubility Spottings Staphylococcus aureus Structure Surface System Tail Time Toxin Transmembrane Transport Trichoderma Tube Tubulin Tubulin Interaction Voltage-Dependent Anion Channel Warburg Effect Work base biological systems botulinum cancer cell carcinogenesis density design deviant driving force hepatoma cell insight interest novel novel strategies particle porin prevent protein protein interaction receptor reconstitution research study respiratory solute sugar syringomycin E tetramethylrhodamine theories two-dimensional uptake

项目摘要

I. Inhalational anesthetic halothane changes the domain structure of a binary lipid membrane The molecular mechanisms of volatile anesthetic action remain obscure despite a long history of research and clinical use for over a century and a half. The remarkable correlation between anesthetic solubility in oil and anesthetic potency, the so-called Meyer-Overton rule, strongly suggests a lipid membrane mediated mechanism. Paradoxically, structural studies of lipid bilayer membranes in the presence of anesthetics yielded negligible effects. In classic experiments 30 years ago employing X-ray and neutron diffraction from dimyristoylphosphatidylcholine (DMPC)/cholesterol membranes, Franks and Lieb found that for inhalation anesthetics ...at surgical concentrations, however, there are no significant changes in bilayer structure. Since then, the conceptual view of cell membranes has shifted from relatively homogeneous lipid bilayers with interspersed proteins to complex lipid mixtures, with laterally separated membrane domains formed as a result of lipid demixing. Accumulating evidence indicates that certain membrane proteins are clustered in domains such as cholesterol-rich lipid rafts. Guided by this conceptual shift, jointly with NIST scientists we performed X-ray and neutron diffraction experiments on a binary lipid membrane. Specifically, we studied a 1:1 mixture of dipalmitoylphosphatidylcholine (DPPC) and dilauroylphaphatidylcholine (DLPC) to demonstrate that halothane, but not dichlorohexafluorocyclobutane, a halogenated nonanesthetic of close properties, produces a pronounced redistribution of lipids between different domains at physiologically relevant concentrations. The domains of different lipid types were identified through their different lamellar d-spacings and isotope composition. These results have demonstrated a specific effect of inhalational anesthetics on mixing phase equilibria. Combined with a growing body of data revealing that conformational dynamics of transmembrane proteins are very sensitive to the parameters of the lipid bilayer within which they reside, our findings suggest that halothane and other volatile anesthetics may act through the cell membrane by changing its domain structure. We hope that these results are of ultimate help in the search for more potent and safe anesthetizing agents. II. VDAC inhibition by tubulin and its physiological implications Recently we have identified an important missing player in the regulation of the Voltage-Dependent Anion Channel (VDAC) of the outer mitochondrial membrane as the abundant cytoskeletal protein tubulin. We have now extended our study of tubulin-VDAC interaction in three directions. First, we studied the structural features of the blocked state as revealed by its sensitivity to the charge of the membrane surface. We found that the relative residual conductance of the tubulin-blocked state of VDAC is sensitive to the surface charge of the membrane thus confirming the tail-in-the-pore model of the blockage suggested by us earlier. Second, we demonstrated that in vitro phosphorylation of VDAC by either glycogen synthase kinase-3beta or cAMP-dependent protein kinase A, increases the on-rate of tubulin binding to the reconstituted channel by orders of magnitude. Experiments on human hepatoma cells HepG2 supported our conjecture that VDAC permeability for the mitochondrial respiratory substrates is regulated by dimeric tubulin and channel phosphorylation. Treatment of HepG2 cells with colchicine prevented microtubule polymerization, thus increasing dimeric tubulin availability in the cytosol. Accordingly, this led to a decrease of mitochondrial potential measured by assessing mitochondrial tetramethylrhodamine methyester uptake with confocal microscopy. Third, we have demonstrated that the mechanism of VDAC blockage by tubulin involves tubulin interaction with the membrane as a critical step. The on-rate of the blockage varies up to 100-fold depending on the particular lipid composition used for bilayer formation in reconstitution experiments. Thus, in addition to revealing an important step in tubulin-VDAC interaction, our results give a new example of the lipid-controlled protein-protein interaction where the choice of lipid species is able to change the equilibrium binding constant by orders of magnitude. Immediate physiological implications of these findings include new insights into cell signaling pathways and cytoskeleton/microtubule activity in health and disease, especially in the case of the highly dynamic microtubule network which is characteristic of carcinogenesis and cell proliferation. These findings may help to identify new mechanisms of mitochondria-associated action of chemotherapeutic microtubule-targeting drugs, and also to understand why and how cancer cells preferentially use inefficient glycolysis rather than oxidative phosphorylation (Warburg effect). III. Physical theory of channel-facilitated transport Further development of the physical theory of channel-facilitated transport is required for deeper understanding of its regulation mechanisms. This year we have concentrated on two topics: the effects of entropy barriers and clustering. Transport in systems of varying geometry that creates entropy barriers in one-dimensional description has become the subject of growing interest among researchers in recent years. We investigated transport of point Brownian particles in a tube formed by identical periodic compartments of varying diameter, focusing on the effects that are due to the compartment asymmetry. In particular, we studied the force-dependent mobility of the particle and found that the mobility is a symmetric non-monotonic function of the driving force when the compartment is symmetric. However, the compartment asymmetry gives rise to an asymmetric force-dependent mobility, which remains non-monotonic when the compartment asymmetry is not too high and becomes monotonic in tubes formed by highly asymmetric compartments. The transition of the dependence from non-monotonic to monotonic behavior results in important consequences for the particle motion under the action of a time-periodic force with zero mean: In a tube formed by moderately asymmetric compartments, the particle under the action of such a force moves with an effective drift velocity that vanishes at small and large values of the force amplitude having a maximum in between. In a tube formed by highly asymmetric compartments, the effective drift velocity monotonically increases with the amplitude of the driving force and becomes unboundedly large as the amplitude tends to infinity. Clustering of receptors, transporters, and ion channels, which seems to be the rule rather than the exception, complicates the qualitative description of transport in biological systems. We have analyzed the effects of clustering by considering an aggregate of absorbing disks on the otherwise reflecting wall. Trapping of diffusing particles by such an aggregate is a manifestly many-body problem because of the disk competition for the particles. By replacing the cluster with an effective uniformly absorbing spot, we derived a simple algebraic expression for the rate constant that characterizes the trapping. The formula shows how the rate constant depends on the size, shape, and the density of packing in the cluster. The obtained analytical results may provide a rationale for surface clustering on the plasma membranes in addition to the usual explanation of enhancing two-dimensional macromolecular interaction.

I.吸入麻醉卤代改变二元脂质膜的结构域结构尽管研究和临床用途有一个半世纪的历史，但挥发性麻醉作用的分子机制仍然晦涩难懂。所谓的Meyer-Overton规则在油中麻醉溶解度与麻醉效力之间的显着相关性强烈暗示了脂质膜介导的机制。矛盾的是，在麻醉药存在下脂质双层膜的结构研究产生了可忽略的作用。在30年前的经典实验中，采用X射线和中子衍射，来自Dimerristoylphospadidyl胆碱（DMPC）/胆固醇膜，Franks和Lieb发现，在手术浓度下吸入麻醉剂，但是，在手术浓度下，Bilayer结构没有重大变化。从那时起，细胞膜的概念视图已从带有散布蛋白质的相对均匀的脂质双层转移到复杂的脂质混合物，由于脂质脱粒而形成的侧向分离的膜结构域。积累的证据表明某些膜蛋白聚集在诸如富含胆固醇的脂质筏等域中。在这种概念上的转变的指导下，我们与NIST科学家共同进行了X射线和中子衍射实验。 Specifically, we studied a 1:1 mixture of dipalmitoylphosphatidylcholine (DPPC) and dilauroylphaphatidylcholine (DLPC) to demonstrate that halothane, but not dichlorohexafluorocyclobutane, a halogenated nonanesthetic of close properties, produces a pronounced redistribution of lipids between different domains at physiologically relevant浓度。不同脂质类型的结构域通过其不同的层状D间隙和同位素组成鉴定。这些结果表明吸入麻醉对混合阶段平衡的特定作用。结合越来越多的数据表明，跨膜蛋白的构象动力学对它们所居住的脂质双层的参数非常敏感，我们的发现表明硫烷和其他挥发性麻醉剂可以通过改变其域结构来通过细胞膜来起作用。我们希望这些结果在寻找更有效和安全的麻醉剂方面有最终的帮助。 ii。 VDAC抑制微管蛋白及其生理意义最近，我们确定了一个重要的缺失参与者在调节线粒体外膜的电压依赖性阴离子通道（VDAC）中是丰富的细胞骨架蛋白微管蛋白。现在，我们已经在三个方向上扩展了小管蛋白-VDAC相互作用的研究。首先，我们研究了阻塞状态的结构特征，如其对膜表面电荷的敏感性所揭示的。我们发现，VDAC的小管蛋白阻滞状态的相对残留电导对膜的表面电荷敏感，从而证实了我们之前提出的阻塞的尾巴模型。其次，我们证明了通过糖原合酶激酶-3BETA或cAMP依赖性蛋白激酶A对VDAC进行体外磷酸化，从而增加了微管蛋白与重构通道的速率增加。人肝癌细胞HEPG2的实验支持我们的猜想，即VDAC对线粒体呼吸道底物的渗透性受二聚体微管蛋白和通道磷酸化调节。用秋水仙碱治疗HEPG2细胞可预防微管聚合，从而增加了细胞质中的二聚体小管蛋白的可用性。因此，这导致通过评估与共聚焦显微镜摄入的线粒体四甲基二胺摄取来测量的线粒体电位的降低。第三，我们证明了微管蛋白的VDAC阻塞机理涉及与膜的相互作用，作为关键步骤。阻塞的速率根据用于重构实验中双层形成的特定脂质组成而变化100倍。因此，除了揭示微管蛋白-VDAC相互作用的重要步骤外，我们的结果还为脂质控制的蛋白质 - 蛋白质相互作用提供了一个新的例子，其中脂质物种的选择能够通过巨大的顺序改变平衡结合常数。这些发现的直接生理意义包括对健康和疾病中细胞信号通路和细胞骨架/微管活性的新见解，尤其是在高度动态的微管网络的情况下，这是癌变和细胞增殖的特征。这些发现可能有助于确定化学治疗微管靶向药物的线粒体相关作用的新机制，并了解为什么癌细胞优先使用无效的糖酵解而不是氧化磷酸化（沃堡效应）。 iii。通道传输的物理理论需要进一步发展渠道相关运输的物理理论，才能更深入地了解其调节机制。今年，我们专注于两个主题：熵屏障和聚类的影响。近年来，在一维描述中创建熵障碍的各种几何系统的运输已成为研究人员日益增长的兴趣的主题。我们研究了点在管道中相同的周期隔室形成的棕色颗粒的运输，该隔室的直径相同，重点是由于腔室不对称而引起的效果。特别是，我们研究了粒子的力依赖性迁移率，发现当隔室是对称的时，迁移率是驱动力的对称非主体函数。然而，室不对称会产生不对称的力依赖性迁移率，当隔室不对称性不太高时，它仍然是非单调的，并且在由高度不对称室形成的试管中变得单调。从非单调行为到单调行为的依赖性过渡会导致粒子运动在零平均值的时间周期性作用下对粒子运动产生重要的后果：在中等不对称的隔室形成的管中，在这种力量的作用下，粒子以有效的漂移速度移动，有效的漂移速度在小小的和较大的力量上消失了，这是相互效果的最大值，具有最大值的质量。在由高度不对称室形成的管中，有效的漂移速度单调地随驱动力的振幅增加而增加，并且随着振幅趋向于无穷大。受体，转运蛋白和离子通道的聚类似乎是规则，而不是例外，这使生物系统中传输的定性描述变得复杂。我们通过考虑在原本反射壁上考虑吸收磁盘的总体来分析聚类的效果。由于粒子的磁盘竞争，这种骨料捕获这种散射颗粒是一个显然是多体问题。通过用一个有效的均匀吸收点代替簇，我们得出了一个简单的代数表达式，以表征捕获的速率常数。该公式显示速率常数如何取决于群集中包装的大小，形状和密度。获得的分析结果还可以为质膜上表面聚类提供基本原理，除了通常的解释增强了二维大分子相互作用。