Extending Chemical Synthesis to Ion Channels and Transporters

描述(申请人提供)：离子通道和转运蛋白是完整的膜蛋白，在人体生理学的几乎所有方面都扮演着重要的角色。了解这些蛋白质的内部工作机制很重要，因为离子通道和转运蛋白的功能障碍会导致人类疾病。在这个提议中，我们使用一种新的方法，化学合成，来研究这些蛋白质。化学合成是一种强有力的蛋白质修饰方法，因为它允许将多种非天然氨基酸和蛋白质骨架修饰结合在一起，以实现蛋白质的精确变化。在这个应用中，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+通道选择性过滤器的构象变化 这会将其从导电状态转换为非导电状态。慢失活在决定可兴奋细胞的电学性质方面起着至关重要的作用。我们将解决以下关于缓慢失活的悬而未决的问题：i)在缓慢失活状态下选择性过滤器的构象是什么？Ii)离子是如何调节缓慢失活的？和iii)在不同K+中缓慢失活的选择性过滤器上是否发生了类似的构象变化 频道？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相结合，并使用X射线结晶学进行结构研究。我们还建议将化学合成扩展到转运体，通过进行GltPH的合成，GltPH是真核细胞谷氨酸转运体的古生代同源物。谷氨酸转运体通过利用离子的电化学梯度能量来调节谷氨酸的集中摄取。谷氨酸转运体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。谷氨酸转运体的一个中心悬而未决的问题是，离子的电化学梯度与谷氨酸摄取的耦合机制。在这里，我们使用化学合成的GltPh来揭示Na+偶联转运的机制。这项研究具有重要意义，因为它将为K+通道和Na+耦合转运中缓慢失活的生理重要过程提供更深层次的机制洞察。此外，本研究还将建立研究完整膜蛋白的化学合成方法学。

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.