Biophysics of Large Membrane Channels

大膜通道的生物物理学

基本信息

批准号：
7594111
负责人：
sergey bezrukov
金额：
$ 71.66万
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/7594111
关键词：
Affect Affinity Amino Acids Anthrax disease Antigens Apoptosis Area Behavior Biological Biological Assay Biophysics Cardiolipins Cell Membrane Permeability Cells Characteristics Chemicals Closure Condition Cytoprotection Drug Design Edema Ethylene Glycols Frequencies Goals Gramicidin Hemolysin Infection Inner mitochondrial membrane Ion Channel Ions Lecithin Length Lipid Bilayers Lipids Mechanics Membrane Membrane Lipids Mitochondria Modeling Molecular Molecular Conformation Nature Numbers Organelles Pathology Phosphatidylethanolamine Physics Play Polymers Probability Process Property Protein Conformation Proteins Pseudomonas syringae Range Regulation Relaxation Reporting Role Series Shapes Side Site Staging Staphylococcal Infections Structure Testing Therapeutic Agents Time Toxin Variant Viral Virulence Factors Voltage-Dependent Anion Channel Water anthrax lethal factor anthrax toxin base beta-Cyclodextrins betadex cytotoxicity drug discovery ethylene glycol gramicidin A inhibitor/antagonist mitochondrial membrane novel therapeutics particle pathogen phosphatidylethanolamine porin programs radius bone structure receptor reconstitution research study residence single molecule size solute syringomycin E theories tool voltage

项目摘要

Large ion channels in cell and organelle membranes are not only the gateways of metabolite exchange between different cellular compartments and cells; they are also recognized as multifunctional membrane receptors and can be formed by components of many toxins. To study these channels under precisely controlled conditions, we purify and then reconstitute channel-forming proteins into planar lipid bilayers. I. High-affinity blocking of anthrax and other toxin channels. Many pathogens use the formation of trans-membrane pores in target cells in the process of infection. A great number of pore-forming proteins, both bacterial and viral, are considered to be important virulence factors. This makes them attractive targets for the discovery of new therapeutic agents. Recently, using structure-inspired drug design, we demonstrated that aminoalkyl derivatives of beta-cyclodextrin inhibited anthrax lethal toxin (LeTx) action by blocking the trans-membrane pore formed by the protective antigen (PA) subunit of the toxin. Now, as a next step, we evaluate a series of new beta-cyclodextrin derivatives with the goal of identifying potent inhibitors of anthrax toxins. Newly synthesized hepta-6-thioaminoalkyl and hepta-6-thioguanidinoalkyl derivatives of beta-cyclodextrin with the alkyl spacers of various lengths were tested for the ability to inhibit cytotoxicity of LeTx in cells as well as to block ion conductance through PA channels reconstituted in planar bilayer lipid membranes. Most of the tested derivatives were protective against anthrax LeTx action at low or sub-micromolar concentrations. They also blocked ion conductance through PA channels at concentrations as low as 0.1 nM. The activity of the derivatives in both cell protection and channel blocking was found to depend on the length and chemical nature of the substituent groups. One of the compounds was also shown to block the edema toxin activity. To test the broader applicability of this approach, we sought beta-cyclodextrin derivatives capable of inhibiting the activity of Staphylococcas aureus alpha-Hemolysin (HL), which is regarded as a major virulence factor playing an important role in staphylococcal infection. We identified several amino acid derivatives of beta-cyclodextrin that inhibited the activity of HL and LeTx in cell-based assays at low micromolar concentrations. One of the compounds was tested for the ability to block ion conductance through the pores formed by HL and PA in artificial lipid membranes. We anticipate that this approach can serve as the basis for a structure-directed drug discovery program to find new and effective therapies against various pathogens that utilize pore-forming proteins as virulence factors. II. Regulation of VDAC by non-lamellar lipids of mitochondrial membranes. Evidence accumulates that lipids play important roles in permeabilization of the mitochondria outer membrane (MOM) at the early stage of apoptosis. Lamellar phosphatidylcholine (PC) and non-lamellar phosphatidylethanolamine (PE) lipids are the major membrane components of the MOM. Cardiolipin (CL), the characteristic lipid from the mitochondrial inner membrane, is another non-lamellar lipid recently shown to play a role in MOM permeabilization. We investigate the effect of these three key lipids on the gating properties of voltage-dependent anion channel (VDAC), the major channel in MOM. We find that PE induces voltage asymmetry in VDAC current-voltage characteristics by promoting channel closure at cis-negative applied potentials. Significant asymmetry is also induced by CL. The observed differences in VDAC behavior in PC and PE membranes cannot be explained by differences in the insertion orientation of VDAC in these membranes. Rather, it is clear that the two non-lamellar lipids affect VDAC gating. Using gramicidin A channels as a tool to probe bilayer mechanics, we show that VDAC channels are much more sensitive to the presence of CL than it could be expected from the experiments with gramicidin channels. We suggest that this is due to the preferential insertion of VDAC into CL-rich domains. We propose that the specific lipid composition of the mitochondria outer membrane and/or of contact sites might influence MOM permeability by regulating VDAC gating. III. Physics of channel-facilitated metabolite transport. The past years progress in quantitative understanding of channel-facilitated transport resulted in the following findings. 1) To probe the size of the ion channel formed by Pseudomonas syringae lipodepsipeptide Syringomycin E, we used the partial blockage of ion current by penetrating poly(ethylene glycol)s. Earlier experiments with symmetric application of these polymers yielded a radius estimate of 1 nm. Now, motivated by the asymmetric non-ohmic current-voltage curves reported for this channel, we explored its structural asymmetry. We gauged this asymmetry by studying the channel conductance after one-sided addition of differently sized poly(ethylene glycol)s. We have found that small polymers added to the cis-side of the membrane (the side of lipodepsipeptide addition) reduce channel conductance much less than do the same polymers added to the trans-side. We interpret these results to suggest that the water-filled pore of the channel is conical with cis- and trans-radii differing by a factor of 23 and that the smaller cis-radius is in the 0.250.35 nm range. In symmetric, two-sided addition, polymers entering the pore from the larger opening dominate blockage. 2) We studied the distribution of direct translocation times for particles passing through membrane channels between two reservoirs. The direct translocation time is a conditional first-passage time defined as the residence time of the particle in the channel while passing through the membrane directly, i.e., without returning to the reservoir from which it entered. We have shown that the distributions of direct translocation times are identical for translocation in both directions, independent of any asymmetry in the potential across the channel and, hence, the translocation probabilities. 3) Channel-forming proteins in a lipid bilayer of a biological membrane usually respond to variation of external voltage by changing their conformations. Periodic voltages with frequency comparable with the inverse relaxation time of the protein produce hysteresis in the occupancies of the protein conformations. If the channel conductance changes when the protein jumps between these conformations, hysteresis in occupancies is observed as hysteresis in ion current through the channel. We have developed an analytical theory of this phenomenon assuming that the channel conformational dynamics can be described in terms of a two-state model. The theory describes transient behavior of the channel after the periodic voltage is switched on as well as the shape and area of the stationary hysteretic loop as functions of the frequency and amplitude of the applied voltage.

细胞和细胞器膜中的大离子通道不仅是不同细胞隔室和细胞之间代谢物交换的网关。它们也被认为是多功能膜受体，可以由许多毒素的成分形成。为了在精确控制的条件下研究这些通道，我们将纯化的蛋白质净化，然后重新构建蛋白质成平面脂质双层。 I.炭疽和其他毒素通道的高亲和力阻断。许多病原体在感染过程中使用靶细胞中跨膜孔的形成。细菌和病毒的大量孔形成蛋白被认为是重要的毒力因子。这使它们成为发现新的治疗剂的吸引人目标。最近，使用结构启发的药物设计，我们证明了β-环糊精的氨基烷基衍生物通过阻断由毒素的保护性抗原（PA）亚基形成的跨膜孔，抑制了炭疽致死的毒素（LETX）作用。现在，作为下一步，我们评估了一系列新的β-环糊精衍生物，目的是识别炭疽毒素的有效抑制剂。新合成的HEPTA-6-硫氨基烷基和Hepta-6-硫代氨基烷基衍生物的β-链霉素的衍生物具有各种长度的烷基间隔剂，以抑制细胞中LETX的细胞毒性在PANELS中抑制LETX的细胞毒性通过PANELS PRAMER praneL praneR praner bliLay byerare bilayer bliLay bliLay bliLay bylay bliLay bliLay。大多数测试的衍生物在低或亚微摩尔浓度下针对炭疽LETX作用具有保护性。他们还以低至0.1 nm的浓度通过PA通道阻止了离子电导。发现衍生物在细胞保护和通道阻塞中的活性取决于取代基团的长度和化学性质。其中一种化合物也显示出可以阻断水肿毒素活性。为了测试这种方法的更广泛的适用性，我们寻求β-环糊精衍生物，能够抑制金黄色葡萄球菌金黄色葡萄球菌α-蛋白酶（HL）的活性，后者被视为主要毒力在葡萄球菌感染中起重要作用。我们确定了β-环糊精的几种氨基酸衍生物，这些氨基酸衍生物在低微摩尔浓度下抑制了基于细胞的测定中HL和LETX的活性。测试了其中一种化合物的能力，能够通过HL和PA在人造脂质膜中形成的孔阻断离子电导。我们预计，这种方法可以作为结构指导的药物发现计划的基础，以发现针对利用孔形成蛋白作为毒力因素的各种病原体的新有效疗法。 ii。通过线粒体膜的非层状脂质对VDAC进行调节。有证据表明，脂质在细胞凋亡的早期阶段在线粒体外膜（MOM）的通透性中起重要作用。层状磷脂酰胆碱（PC）和非层状磷脂酰乙醇胺（PE）脂质是MOM的主要膜成分。 Cardiolipin（Cl）是线粒体内膜的特征性脂质，是最近显示出的另一种非层状脂质，它在MOM透化中起作用。我们研究了这三个关键脂质对电压依赖性阴离子通道（VDAC）的门控性能的影响，这是MOM的主要通道。我们发现，PE通过在顺式阴性应用电位上促进通道闭合来引起VDAC电流 - 电压特性的电压不对称性。 CL也诱导了明显的不对称性。在PC和PE膜中观察到的VDAC行为的差异无法通过这些膜中VDAC插入方向的差异来解释。相反，很明显，两种非层状脂质会影响VDAC门控。使用gramicidin A通道作为探测双层力学的工具，我们表明VDAC通道对CL的存在要比使用Gramicidin通道的实验所期望的要敏感得多。我们建议这是由于将VDAC插入富含Cl的域而造成的。我们建议线粒体外膜和/或接触位点的特异性脂质组成可能通过调节VDAC门控而影响MOM的渗透性。 iii。通道促进代谢物转运的物理。过去几年对渠道相关运输的定量理解的进展导致了以下发现。 1）为了探测由丁香脂蛋白酶肌霉素E形成的离子通道的大小，我们通过穿透聚（乙烯乙二醇）s使用了离子电流的部分阻塞。这些聚合物的对称应用的早期实验得出的半径估计值为1 nm。现在，由该通道报道的不对称非欧马电流 - 电压曲线的动机，我们探索了其结构不对称性。我们通过研究不同尺寸的聚（乙二醇）s后研究通道电导来测量这种不对称性。我们发现，添加到膜的顺式侧面（脂肽添加的侧面）降低了通道电导率的降低远小于添加到跨侧的相同聚合物的降低得多。我们将这些结果解释为表明该通道的水孔是圆锥形的，顺式和反式radii的孔不同于23倍，并且较小的顺式 - 拉迪乌斯在0.250.35 nm范围内。在对称的，双面的添加中，聚合物从较大的开口占主导地位进入孔。 2）我们研究了穿过两个储层之间膜通道的颗粒的直接转运时间的分布。直接易位时间是一个有条件的第一学期时间，定义为粒子在通道中的停留时间，同时直接通过膜，即，而无需返回进入其进入的储层。我们已经表明，直接易位时间的分布在这两个方向上都是相同的，与整个通道电位的任何不对称性无关，因此与易位概率相同。 3）生物膜的脂质双层中的通道形成蛋白通常通过改变其构象来应对外部电压的变化。与蛋白质构象占用物相当的频率的周期性电压与蛋白质的逆松弛时间相当。如果当蛋白在这些构象之间跳跃时，通道电导变化，则观察到占用率的滞后是通过通道在离子电流中的滞后。假设可以用两态模型来描述通道构象动力学，我们已经开发了这种现象的分析理论。该理论描述了在打开周期电压后的通道的瞬态行为以及固定滞后环的形状和面积作为施加电压的频率和振幅的函数。