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

基本信息

批准号：
8921212
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
项目类别：
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/8921212
关键词：
Action Potentials Address Alzheimer&apos s Disease Amino Acids Amyotrophic Lateral Sclerosis Aspartate Binding Binding Sites Biological Process Biology Cells Coupled Coupling Electrophysiology (science)Family Functional disorder Glutamate Transporter Glutamates Goals Grant Health 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 cardiac pacing chemical synthesis electrical property human disease insight interdisciplinary approach member nervous system disorder novel strategies protein structure protein structure function tool uptake

项目摘要

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在人类生理学的几乎所有方面中起重要作用的整体膜蛋白。了解这些蛋白质的内部起作用很重要，因为离子通道和转运蛋白的功能障碍会导致人类疾病。在此提案中，我们使用一种新颖的方法，即化学合成，研究这些蛋白质。化学合成是蛋白质修饰的强大方法，因为它允许掺入各种不自然的氨基酸和蛋白质骨架修饰，以进行蛋白质的精确变化。在此应用中，我们使用KCSA和KVAP的K+通道的化学合成来研究缓慢失活的机理。慢速失活是K+通道的选择性滤波器的构象变化这将其从导电态转换为非导电状态。缓慢的失活在确定可激发细胞的电性能中起着至关重要的作用。我们将解决有关缓慢失活的以下未解决的问题：i）在缓慢失活的状态下，选择性的构象是什么？ ii）渗透离子如何调节缓慢的失活？ iii）在选择性滤波器上进行类似的构象变化是不同K+慢速失活的基础频道？我们将采用一种多学科的方法进行这些研究，该方法将化学合成与电生理学和使用X射线晶体学研究结合在一起。我们还建议通过执行真核谷氨酸转运蛋白的古细菌同源物GLTPH的合成来扩展化学合成至转运蛋白。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浓度吸收。谷氨酸转运蛋白的功能障碍与阿尔茨海默氏症和肌萎缩性侧索硬化症（ALS）等神经系统疾病有关。谷氨酸转运蛋白中的一个中心未解决的问题是离子的电化学梯度与谷氨酸摄取的机制。在这里，我们使用GLTPH的化学合成来阐明Na+耦合转运的机理。提出的研究很重要，因为它将为在K+通道和Na+耦合转运的生理重要过程中提供更深入的机理见解。此外，这项研究将建立用于研究整体膜蛋白的化学合成方法。