Extending Chemical Synthesis to Ion Channels and Transporters

将化学合成扩展到离子通道和转运蛋白

• 批准号: 8758500
• 负责人: Francis Valiyaveetil
• 金额: $ 30.8万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8758500
• 关键词: Action Potentials; Address; Alzheimer' s Disease; Amino Acids; Amyotrophic Lateral Sclerosis; Aspartate; Binding; Binding Sites; Biological Process; Biology; Cardiac; Cells; Coupled; Coupling; Electrophysiology (science); Family; Functional disorder; Glutamate Transporter; Glutamates; Goals; Grant; Homologous Gene; Human; Integral Membrane Protein; Investigation; Ion Channel; Ions; Knowledge; Lead; Light; Link; Mediating; Methodology; Methods; Modification; Molecular Conformation; Neurons; Oxygen; Physiology; Play; Post-Translational Protein Processing; Potassium Channel; Process; Protein Family; Proteins; Research; Role; Site; Site-Directed Mutagenesis; Structure; Techniques; Vertebral column; Work; X-Ray Crystallography; chemical synthesis; electrical property; human disease; insight; interdisciplinary approach; member; nervous system disorder; novel strategies; protein structure; protein structure function; public health relevance; tool; uptake

DESCRIPTION (provided by applicant): Ion channels and transporters are integral membrane proteins that play important roles in virtually all aspects of human physiology. Understanding the inner workings of these proteins is important, as dysfunctions of ion channels and transporters lead to human diseases. In this proposal, we use a novel approach, chemical synthesis, to investigate these proteins. Chemical synthesis is a powerful method for protein modification because it allows the incorporation of a wide variety of unnatural amino acids and protein backbone modifications for precise changes in the protein. In this application, we use the chemical synthesis of the K+ channels, KcsA and KvAP, to investigate the mechanism of slow inactivation. Slow inactivation is a conformational change at the selectivity filter of K+ channels that converts it from a conductive to a non- conductive state. Slow inactivation plays a crucial role in determining the electrical properties of an excitable cell. We will address the following unresolved issues regarding slow inactivation: i) What is the conformation of the selectivity filte in the slow inactivated state? ii) How do permeant ions modulate slow inactivation? and iii) Do similar conformational changes at the selectivity filter underlie slow inactivation in different K+ channels? We will employ a multidisciplinary approach for these investigations that combines chemical synthesis with electrophysiology and structural studies using X-ray crystallography. We also propose to extend chemical synthesis to transporters by carrying out the synthesis of GltPH, an archaeal homolog of eukaryotic glutamate transporters. Glutamate transporters mediate the concentrative uptake of glutamate by harnessing the energy from the electrochemical gradient of ions. Dysfunction of glutamate transporters has been implicated in neurological diseases such as Alzheimer's and amyotrophic lateral sclerosis (ALS). A central unanswered question in glutamate transporters is the mechanism by which the electrochemical gradients of the ions are coupled to the uptake of glutamate. Here we use chemical synthesis of GltPH to unravel the mechanism of Na+ coupled transport. The research proposed is significant because it will provide deeper mechanistic insights into the physiologically important processes of slow inactivation in K+ channels and Na+ coupled transport. Further, this research will establish the methodology of chemical synthesis for investigating integral membrane proteins.

描述（由申请人提供）：离子通道和转运蛋白是完整的膜蛋白，在人类生理学的几乎所有方面都发挥着重要作用。了解这些蛋白质的内部运作非常重要，因为离子通道和转运蛋白的功能障碍会导致人类疾病。在这个提案中，我们使用一种新的方法，即化学合成，来研究这些蛋白质。化学合成是一种强大的蛋白质修饰方法，因为它允许掺入多种非天然氨基酸和蛋白质主链修饰，以精确改变蛋白质。在此应用中，我们使用 K+ 通道 KcsA 和 KvAP 的化学合成来研究缓慢失活的机制。缓慢失活是 K+ 通道选择性过滤器处的构象变化 将其从导电状态转换为非导电状态。缓慢失活在确定可兴奋细胞的电特性方面起着至关重要的作用。我们将解决以下有关慢速灭活的未解决问题： i) 慢速灭活状态下选择性过滤器的构象是什么？ ii) 渗透离子如何调节缓慢失活？ iii) 在选择性过滤器上是否有类似的构象变化导致不同 K+ 中的缓慢失活 渠道？我们将采用多学科方法进行这些研究，将化学合成与电生理学以及使用 X 射线晶体学的结构研究相结合。我们还建议通过合成 GltPH（真核谷氨酸转运蛋白的古菌同源物）来将化学合成扩展到转运蛋白。谷氨酸转运蛋白通过利用离子电化学梯度的能量来介导谷氨酸的集中摄取。谷氨酸转运蛋白功能障碍与阿尔茨海默病和肌萎缩侧索硬化症 (ALS) 等神经系统疾病有关。谷氨酸转运蛋白中一个尚未解答的核心问题是离子的电化学梯度与谷氨酸的摄取耦合的机制。在这里，我们利用 GltPH 的化学合成来揭示 Na+ 耦合运输的机制。这项研究意义重大，因为它将为 K+ 通道缓慢失活和 Na+ 耦合运输的重要生理过程提供更深入的机制见解。此外，这项研究将建立研究完整膜蛋白的化学合成方法。