Extending Chemical Synthesis to Ion Channels and Transporters

描述（由申请人提供）：离子通道和转运体是完整的膜蛋白，在人体生理的几乎所有方面都起着重要作用。了解这些蛋白质的内部工作原理很重要，因为离子通道和转运体的功能失调会导致人类疾病。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。化学合成是一种强大的蛋白质修饰方法，因为它允许将各种非天然氨基酸和蛋白质主链修饰结合在一起，以精确地改变蛋白质。在这个应用中，我们使用化学合成的K+通道，KcsA和KvAP，来研究缓慢失活的机制。缓慢失活是K+通道选择性滤光器的构象变化

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.