Molecular Dissection of the Permeability Transition Pore

渗透率转变孔的分子解剖

• 批准号: 7214064
• 负责人: MICHAEL A FORTE
• 金额: $ 29.58万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7214064
• 关键词: Adenine Nucleotides; Affinity; Apoptosis; Apoptotic; Attention; BCL-2 Protein; BCL2 gene; Bioenergetics; Biological; Biological Assay; Cell Death; Cell Membrane Permeability; Cell Survival; Cells; Charge; Chemicals; Complex; Cyclosporine; Data; Development; Diagnostic; Disease; Dissection; Energy Metabolism; Event; Family; Family member; Goals; Homeostasis; Human; Immunosuppressive Agents; In Vitro; Inner mitochondrial membrane; Knock-out; Laboratories; Lead; Life; Maintenance; Mediator of activation protein; Membrane; Membrane Potentials; Metabolism; Mitochondria; Modeling; Modification; Molecular; Multiprotein Complexes; Mus; Mutant Strains Mice; Nerve Degeneration; Outer Mitochondrial Membrane; Participant; Pathologic Processes; Permeability; Play; Process; Production; Protein Isoforms; Proteins; Proton Pump; Protons; Reagent; Reperfusion Injury; Respiration; Ro 68-3400; Role; Rupture; Site; Standards of Weights and Measures; Structure; System; Testing; Tissues; VDAC1 gene; Yeasts; analog; base; cell growth regulation; cyclophilin D; cytochrome c; experience; genetic regulatory protein; human disease; in vivo; inhibitor/antagonist; mitochondrial dysfunction; mitochondrial permeability transition pore; molecular mass; novel; novel strategies; programs; pyridine nucleotide; research study; respiratory; solute; tool

Mitochondria playa pivotal role in cell survival and tissue development by virtue of their role in energy metabolism, regulation of cellular Ca2 + homeostasis and apoptosis. Given this multifactorial role, they regulate cellular Ca2+ metabolism and bioenergetics function as an integrated s~stem. In terms of normal physiology, this integration is reflected in mitochondrion's high capacity to store Ca2 +, which may protect cells like neurons against transient elevation in intracellular Ca2 + during periods of hyperactivity. Mitochondrial Ca2 + homeostasismust be tightly regulated and is based in a series of specific uptake and release systems. Yet, in vitro themitochondrial inner membrane (IMM) can easily undergo a permeability increase to solutes with molecularmasses of about 1,500 Da or lower. This permeability change, called the permeability transition (PT), isregulated by the opening of a membrane pore, the mitochondrial permeability transition pore (PTP). The PTPis voltage-dependent, cyclosporin A (CsA)-sensitive, high-conductance channel of the inner mitochondrialmembrane; pore open-closed transitions are highly regulated by multiple effectors that likely converge on asmaller set of regulatory sites. The PTP has long been implicated as a target for mitochondrial dysfunction invivo, particularly in the context of specific human pathological events. These suspicions have been confirmedby examination of mice in which the expression of mitochondrial CyPD (a key regulator of PTP action and thetarget of CsA) has been eliminated. These studies have confirmed a critical role for the PTP in models of ischemia/reperfusion injury both in the heart and the brain, models of muscular dystrophy, in the axonaldamage occurring during MS, and Alzheimer's disease. However, despite detailed functional characterizationover the last 30 years, none of the candidate pore components in traditional models has withstood critical andunambiguous genetic tests. In this light, the PBR remains the only biochemically identified component in traditional molecular models of the PTP that has not been subjected to thorough genetic testing. Consequently, the overall goal of this application is to use biochemical and genetic tools to critically test the role of the peripheral benzodiazepine receptor (PBR) in PTP function using a variety of in vitro and in vivo tests that we have developed to confirm its role. either as core components or regulators of the PTP. Our studies are based in mice that we have now successfully generated in which the wild-type Tspo gene has been replaced with a modified Tspo gene containing /oxP sites. Using these mice, our plan is to test PTP function in mitochondria, cells and tissues lacking the PBR and thereby rigorously evaluate the role of the PBR in PTP function. Importantly. since mice have now been successfully generated containing a modified Tspo gene containing loxP sites. it is reasonable that we will be able to complete these aims in the two years of funding provided.

线粒体通过其在细胞存活和组织发育中的作用在细胞存活和组织发育中发挥关键作用 能量代谢、细胞内钙稳态调节和细胞凋亡。考虑到这一多因素 它们的作用是调节细胞内的钙代谢，而生物能量学的作用就像一个完整的S干。在……里面 就正常生理而言，这种整合反映在线粒体存储钙的高能力上 +，这可能保护神经元等细胞 对抗细胞内钙离子在多动期的一过性升高。线粒体钙离子 体内稳定期必须受到严格的监管，并建立在一系列特定的摄取和释放系统的基础上。 然而，在体外，线粒体内膜(Imm)很容易经历通透性增加到 分子量约为1,500 Da或以下的溶质。这种渗透性的变化被称为 通透性转换(PT)，由膜孔、线粒体的开放来调节 渗透率转换孔(PTP)。PTP是电压依赖的，对环孢素A(CsA)敏感， 线粒体膜内的高电导通道；孔的开闭过渡 受到多个效应器的高度调控，这些效应器可能聚集在一组较小的调控部位上。这个 长期以来，PTP一直被认为是体内线粒体功能障碍的靶点，特别是在 特定人类病理事件的背景。这些怀疑已被检验证实。 线粒体CyPD(PTP作用的关键调节因子和靶标)的表达 CsA)已被淘汰。这些研究证实了PTP在动物模型中的关键作用。 心脏和脑的缺血/再灌注损伤，肌营养不良的模型，在 在多发性硬化症和阿尔茨海默病期间发生的轴突损伤。然而，尽管有详细的功能 在过去的30年里，传统模型中的候选孔隙成分 经受住了关键而明确的基因测试。在这一点上，PBR仍然是唯一 生物化学鉴定的成分 传统的PTP分子模型还没有经过彻底的基因检测。 因此，这个应用程序的总体目标是使用生化和遗传工具来关键 使用多种药物检测外周苯二氮卓受体(PBR)在PTP功能中的作用 体外和体内试验，我们已经开发，以确认其作用。作为核心组件或 PTP的监管机构。我们的研究是基于我们现在已经成功培育的小鼠身上 野生型TSPO基因已被替换为 修饰的含有/oxP位点的TSPO基因。使用这些小鼠，我们的计划是测试PTP功能 缺乏PBR的线粒体、细胞和组织，从而严格评估PBR在 PTP功能。重要的是。由于现在已经成功地产生了含有经过修饰的 含有loxP位点的TSPO基因。我们能够在这两个方面实现这些目标是合理的 多年来提供的资金。