Biophysics of Large Membrane Channels

大膜通道的生物物理学

• 批准号: 8351086
• 负责人: sergey bezrukov
• 金额: $ 101.9万
• 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/8351086
• 关键词: Actins; Alamethicin; Amino Acid Sequence; Amino Acids; Area; Binding; Biophysics; Cardiomyopathies; Cell membrane; Cells; Characteristics; Charge; Classification; Clostridium botulinum; Clostridium perfringens; Clostridium perfringens epsilon toxin; Code; Complex; Cultured Cells; Cystic Fibrosis; Cytoskeleton; Cytosol; Development; Epilepsy; Escherichia coli; Eukaryotic Cell; Exclusion; Exotoxins; Explosion; Family; Genes; Genome; Goals; Hemolysin; Human Genome; In Vitro; Individual; Intoxication; Ion Channel; Ions; Lipid Bilayers; Mammalian Cell; Mediating; Membrane; Membrane Lipids; Mitochondria; Molecular; Molecular Conformation; Molecular Weight; Muscular Dystrophies; Nucleotides; Nutrient; Organelles; Organism; Outer Mitochondrial Membrane; Pathology; Physiological; Polymers; Protein Isoforms; Proteins; Pseudomonas aeruginosa; Pseudomonas syringae; Radial; Regulation; Residual state; Respiration; Role; Sodium Chloride; Staphylococcus aureus; Surface; System; Toxin; Trichoderma; Tubulin; Tubulin Interaction; Vesicle; Voltage-Dependent Anion Channel; Work; anthrax lethal factor; anthrax protective factor; base; beta-Cyclodextrins; betadex; body system; cell type; design; enzyme activity; in vivo; inhibitor/antagonist; interest; novel strategies; organic base; pathogenic bacteria; porin; pressure; protein function; rapid growth; receptor binding; receptor mediated endocytosis; reconstitution; research study; solute; sugar; syringomycin E; theories; uptake; voltage

To investigate large ion channels under precisely controlled conditions, we first isolate the channel-forming proteins from their host organisms, purify, and then reconstitute them into planar lipid membranes. Our main goal is to elucidate the physical principles and molecular mechanisms responsible for metabolite flux regulation under normal and pathological conditions. The channel-forming proteins of interest include Voltage-Dependent Anionic Channel from the outer membrane of mitochondria (VDAC), B-components of Clostridium botulinum C2 and Clostridium perfringens iota toxins, Bacillus anthracis protective antigen, Clostridium perfringens Epsilon toxin, Escherichia coli general porin OmpF and sugar-specific LamB, Staphylococcus aureus alpha-Hemolysin, Pseudomonas aeruginosa OprF, Trichoderma viride Alamethicin, and Pseudomonas syringae lipopeptide toxin Syringomycin E. I. Tailor-made beta-cyclodextrins as broad-spectrum inhibitors of binary pore-forming exotoxins. Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin are binary exotoxins, which ADP-ribosylate actin in the cytosol of mammalian cells and thereby destroy the cytoskeleton. C2 and iota toxins consist of two individual proteins, an enzymatic active (A-) component and a separate receptor binding and translocation (B-) component. The latter forms a complex with the A-component on the surface of target cells and, after receptor-mediated endocytosis, it mediates the translocation of the A-component from acidified endosomal vesicles into the cytosol. To this end, the B-components form heptameric pores in endosomal membranes, which serve as translocation channels for the A-components. We have demonstrated that a 7-fold symmetrical positively charged beta-cyclodextrin derivative, per-6-S-(3-aminomethyl)benzylthio-beta-cyclodextrin, protects cultured cells from intoxication with C2 and iota toxins in a concentration-dependent manner starting at low micromolar concentrations. We established that the compound inhibited the pH-dependent membrane translocation of the A-components of both toxins in intact cells. Consistently, the compound effectively blocked transmembrane channels formed by the B-components of C2 and iota toxins in planar lipid bilayers in vitro in reconstitution experiments. With C2 toxin, we consecutively ruled out all other possible inhibitory mechanisms showing that the compound did not interfere with the binding of the toxin to the cells or with the enzyme activity of the A-component. In our previous work, this beta-cyclodextrin derivative was identified as one of the most potent inhibitors of the binary lethal toxin of Bacillus anthracis both in vitro and in vivo, implying that it might represent a broad-spectrum inhibitor of binary pore-forming exotoxins from pathogenic bacteria. II. Regulation of voltage-dependent anionic channel from the outer membrane of mitochondria by dimeric tubulin. In our previous work we have shown that one of the most abundant proteins in the cytosol of the majority of eukaryotic cells, dimeric tubulin, is a potent inhibitor of the voltage-dependent anion channel, VDAC, from the outer mitochondrial membrane. The tubulin-VDAC interaction is seen as reversible transitions of the channel, reconstituted into planar lipid membranes, between its open and tubulin-blocked states. Experiments with isolated mitochondria demonstrated that VDAC-tubulin interaction is functionally important in regulation of mitochondrial respiration. The tubulin-blocked state is still highly ion-conductive (about 40% of the open state conductance in 1 M KCl), which may imply that VDAC inhibition by tubulin is limited by the value of this residual conductance. It is believed, however, that the major role of VDAC is regulation of ATP/ADP exchange and not of the flux of small ions, so what is really important is the effect of tubulin blockage on the nucleotide transport. To assess the functional features of the tubulin-blocked state, we have applied three approaches. We first estimated the change in the characteristic radius of VDAC upon its blockage by tubulin using polymer partitioning into the channel in both open and blocked states. Based on the characteristic molecular weight of the polymer that separates partitioning from exclusion, we concluded that the effective cross-sectional area of the channel is reduced by a factor of two as a result of the blockage. Second, we analyzed the blockage-induced change in the channel small-ion selectivity at salt concentrations close to physiological. We showed that selectivity of the channel reverses its sign: from predominantly anionic selectivity in the open state it shifts to cationic selectivity in the tubulin-blocked one. Third, we estimated ATP partitioning into both open and tubulin-blocked channel. We found that while in the open state the addition of ATP reduces channel conductance, it does not change the conductance of the tubulin-blocked state. We concluded that ATP electrostatically and, at least partially sterically, is excluded from the tubulin-blocked state of VDAC thus establishing the functional role of the VDAC-tubulin interaction in regulation of mitochondrial respiration. III. Physical theory of facilitated metabolite transport: A functional role for transporter isoforms. Many of the nutrients that a cell needs for its functioning, such as sugars, amino acids, nucleotides or organic bases, require specialized transporters to cross the cell membrane. The rapid growth of available information, recently characterized as transporter explosion, has led to creation of the transporter classification system, with division of all transporters into channels and carriers. This year we have focused on the physical principles of optimization of carrier-facilitated transport. In the Human Genome there are 43 distinct families of transport systems that comprise more than 300 isoforms of individual solute carriers. Although the majority of these transport systems is responsible for uptake of specific substrates, a substantial number of transporters are used for uptake of the same solute, and often have an overlapping expression of multiple isoforms that exists in the same cell type. So, the naturally arising question is Why are there so many transporter isoforms?. We offer a possible answer to this question by analyzing the carrier-facilitated transport with the focus on the optimal efficiency of the transporter. We demonstrated that at lower substrate concentrations stronger substrate binding is required, and that the deviations from optimal interaction become more critical as the substrate concentration increases, i.e., higher concentrations necessitate more precise tuning. Thus, uniporters designed to transport the same molecule in the same cell have to be optimized with different amino-acid sequences, with one gene coding for a uniporter protein that functions most efficiently at high solute concentrations, while another gene coding for the one that is most efficient at low concentrations. The existence of multiple transporter isoforms that carry the same molecule is well documented for almost any important substrate. Though this variety of isoforms may seem redundant and, in principle, could be explained by the lack of strong evolutionary pressures to decrease the size of the genome, our analysis now offers a different possibility. We have shown that transporter efficiency is fine-tuned to specific ranges of substrate concentration, so that different isoforms might be tailored accordingly, in order to adjust their amino acid composition for the optimal strength of substrate/transporter interactions and the transition rates between different conformations.

为了研究在精确控制条件下的大离子通道，我们首先将河道形成的蛋白从其宿主有机体分离，然后纯化，然后将其重新构成平面脂质膜。 我们的主要目标是阐明在正常和病理条件下负责代谢物调节的物理原理和分子机制。 The channel-forming proteins of interest include Voltage-Dependent Anionic Channel from the outer membrane of mitochondria (VDAC), B-components of Clostridium botulinum C2 and Clostridium perfringens iota toxins, Bacillus anthracis protective antigen, Clostridium perfringens Epsilon toxin, Escherichia coli general porin OmpF and sugar-specific羔羊，金黄色葡萄球菌α-蛋白酶，铜绿假单胞菌OPRF，Trichoderma viride alamethicin和pseudomomonas sirringae lipopopeptide dexeptide doxeptide symymysycin E. I.量身定制的β-环糊精作为二元孔形成外毒素的宽光谱抑制剂。 肉毒乳梭菌C2毒素和灌注梭状芽胞杆菌IOTA毒素是二元外毒素，它们在哺乳动物细胞的胞质醇中aDP-核糖基化肌动蛋白，从而破坏细胞骨架。 C2和IOTA毒素由两种单独的蛋白质组成，一种酶活性（A-）成分以及单独的受体结合和易位（B-）成分。 后者与靶细胞表面的A组分形成了一个复合物，并且在受体介导的内吞作用后，它介导了A组分从酸化的内体囊泡转移到细胞质中。 为此，B组分形成了内体膜中的Heptameric毛孔，该毛孔是A型组件的易位通道。 我们已经证明，一个7倍对称的带电的β-环糊精衍生物PER-6-S-（3-氨基甲基甲基）苯甲酰噻吩 - 贝泰 - 贝泰 - cyclodextrin可保护培养的细胞在低微级浓度下以浓度依赖性的浓度依赖于C2和IOTA毒素在低微级浓度下与IOTA毒素中的中毒。 我们确定该化合物抑制了完整细胞中两种毒素的A-兼容性的pH依赖性膜易位。 一致地，化合物有效地阻断了由平面脂质双层中C2和IOTA毒素在重建实验中在平面脂质双层中形成的跨膜通道。 使用C2毒素，我们连续排除了所有其他可能的抑制作用机制，表明该化合物不会干扰毒素与细胞的结合或与A型成分的酶活性。 在我们以前的工作中，这种β-环糊精衍生物被确定为体外和体内二型致死性炭疽杆菌二元毒素最有效的抑制剂之一，这意味着它可能代表了植物性遗传性细菌的二元孔形成外毒素的广谱抑制剂。 ii。通过二聚小管从线粒体外膜调节电压依赖性阴离子通道。 在我们以前的工作中，我们已经表明，大多数真核细胞二聚体微管蛋白的细胞质中最丰富的蛋白质之一是电压依赖性阴离子通道VDAC的有效抑制剂，来自外部线粒体外膜的VDAC。 微管蛋白-VDAC相互作用被视为通道的可逆过渡，并在其开放蛋白和微管蛋白阻滞状态之间重构为平面脂质膜。 分离的线粒体实验表明，VDAC-微管蛋白相互作用在调节线粒体呼吸方面具有功能重要。微管蛋白阻滞状态仍然是高离子传导性的（约占1 M KCl的开放状态电导的40％），这可能意味着微管蛋白抑制VDAC受到该残留电导率的值的限制。 但是，人们认为VDAC的主要作用是调节ATP/ADP交换，而不是小离子的通量，因此真正重要的是微管蛋白阻塞对核苷酸转运的影响。 为了评估微管蛋白阻滞状态的功能特征，我们采用了三种方法。 我们首先估计了VDAC通过小管蛋白阻塞的特征半径的变化，使用聚合物在开放状态和阻塞状态下将聚合物分配到通道中。 基于将分配与排除分开的聚合物的特征分子量，我们得出结论，该通道的有效横截面区域由于阻塞而减少了两倍。 其次，我们分析了盐浓度接近生理的盐浓度下的阻塞诱导的小离子选择性的变化。 我们表明，通道的选择性逆转了它的符号：从开放状态下的主要阴离子选择性，它转移到微管蛋白阻断中的阳离子选择性。 第三，我们估计ATP分配到开放蛋白和微管蛋白阻滞通道中。 我们发现，在开放状态下，ATP的添加减少了通道电导，但它不会改变微管蛋白阻滞状态的电导。 我们得出的结论是，ATP静电和至少部分在空间上被排除在VDAC的小管蛋白障碍状态之外，从而确立了VDAC-微管蛋白相互作用在调节线粒体呼吸中的功能作用。 iii。促进代谢物转运的物理理论：转运蛋白同工型的功能作用。 细胞需要其功能的许多营养素，例如糖，氨基酸，核苷酸或有机碱，需要专门的转运蛋白才能越过细胞膜。 最近以转运蛋白爆炸为特征的可用信息的快速增长导致了转运蛋白分类系统的创建，并将所有转运蛋白分为渠道和载体。 今年，我们专注于运输运输运输的优化的物理原理。 在人类基因组中，有43个不同的运输系统家族构成了300多个单个溶质载体的同工型。 尽管这些运输系统的大多数负责摄取特定底物，但使用大量转运蛋白用于摄取相同的溶质，并且通常具有在同一细胞类型中存在的多种同工型的重叠表达。 因此，自然出现的问题是为什么有如此多的运输蛋白同工型？ 我们通过分析载体相关的运输，重点关注运输蛋白的最佳效率，从而为这个问题提供了一个可能的答案。 我们证明，在较低的底物浓度下，需要更强的底物结合，并且随着底物浓度的增加，最佳相互作用的偏差变得更加至关重要，即较高浓度需要更精确的调整。 因此，必须用不同的氨基酸序列对同一细胞中同一分子进行相同分子的单位仪进行优化，一个基因编码为单位蛋白，该蛋白在高溶质浓度下最有效地发挥作用，而另一种基因编码是在低浓度下最有效的基因。 对于几乎所有重要的底物，都有充分的文献记录了携带相同分子的多个转运蛋白同工型的存在。 尽管这种多种同工型似乎似乎是多余的，并且原则上可以通过缺乏强大的进化压力来降低基因组的大小来解释，但我们的分析现在提供了不同的可能性。 我们已经表明，转运蛋白的效率对底物浓度的特定范围进行了微调，因此可以相应地定制不同的同工型，以调整其氨基酸组成，以获得底物/转运蛋白相互作用的最佳强度以及不同构型之间的过渡速率。