Probing functional mechanisms in K+ channels using unnatural mutagenesis

利用非自然诱变探索 K 通道的功能机制

• 批准号: 10000155
• 负责人: Francis Valiyaveetil
• 金额: $ 33.1万
• 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 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10000155
• 关键词: Action Potentials; Amides; Amino Acids; Arrhythmia; Binding Sites; Brain; Cardiac; Charge; Chemicals; Complement; Coupled; Crystallography; Cyclic Nucleotides; Disease; Electrophysiology (science); Epilepsy; Esters; Family; Goals; Heart; Homologous Gene; Human; Hydrogen; Hydrogen Bonding; Investigation; Ion Channel; Ions; Knowledge; Membrane Potentials; Membrane Proteins; Methods; Modification; Molecular Conformation; Movement; Mutagenesis; Mutation; Pattern; Phase; Physiological; Physiology; Play; Post-Translational Protein Processing; Potassium Channel; Process; Proteins; Recovery; Research; Role; Side; Site-Directed Mutagenesis; Structure; Vertebral column; Voltage-Gated Potassium Channel; Xenopus oocyte; arginyllysine; cyclic-nucleotide gated ion channels; heart function; insight; interdisciplinary approach; therapeutic development; therapeutic target; unnatural amino acids; voltage; voltage gated channel

Voltage gated K+ (Kv) channels couple the flux of K+ to the membrane potential and play key roles in the brain and heart. Mutations in Kv channels can cause severe diseases in humans such as epilepsies and cardiac arrhythmias. There have been major advances in the structure determination of Kv channels. In spite of the structural information available, there are major questions on the functional mechanisms in Kv channels that remain unanswered. Here we investigate the processes of voltage gating and C-type inactivation that regulate the flux of K+ through Kv channels. We use a multidisciplinary approach centered on unnatural amino acid (UAA) mutagenesis in our investigations. UAA mutagenesis is a very powerful method for protein modification, compared to traditional mutagenesis, because it allows a large variety of side chain modifications and also permits the modification of the protein backbone. We use this approach to investigate the role of the main chain H-bonds in the fourth transmembrane helix (TM4) in voltage gating of the Shaker K+ channel and the hyperpolarization activated and cyclic nucleotide gated ion channel HCN (aim 1). We investigate the role of ion binding sites in the selectivity filter of the Shaker channel in C-type inactivation and we complement the functional studies on Shaker with structural studies on the KvAP channel, an archaeal homolog of the Shaker channel (aim 2). We also investigate the mechanism of C-type inactivation in the hERG K+ channel, which has interesting functional differences from C-type inactivation in the Shaker channel and is physiologically critical for normal cardiac function (Aim 3). The research proposed is significant as it provides greater insight into the functional mechanisms of voltage gating and C-type inactivation in Kv channels. The research is also significant as it will provide a general strategy for using UAA mutagenesis to investigate the role of main chain H-bonds and ion binding sites, which are important for function in many families of membrane proteins.

电压门控性K+（Kv）通道将K+通量耦合到膜电位，并发挥关键作用。 在大脑和心脏中的作用。Kv通道的突变可导致人类严重疾病 例如癫痫和心律失常。在结构上有了很大的进步 Kv通道的确定。尽管有可用的结构信息， 关于KV通道的功能机制的问题仍然没有答案。这里我们 研究了电压门控和C型失活对K+通量的调节作用 通过Kv频道我们使用多学科的方法，以非天然氨基酸为中心， (UAA)在我们的研究中，UAA诱变是一种非常有效的方法， 蛋白质修饰，与传统的诱变相比，因为它允许大量的各种各样的突变。 侧链修饰，并且还允许蛋白质骨架的修饰。我们用这个 一种研究第四跨膜螺旋中主链氢键作用的方法 (TM4)在Shaker K+通道的电压门控和超极化激活和循环中， 核苷酸门控离子通道HCN（aim 1）。我们研究了离子结合位点的作用， 选择性过滤器的振荡器通道在C型失活，我们补充功能 对Shaker的研究以及对KvAP通道的结构研究，KvAP通道是一种古细菌同源物， 振动器通道（目标2）。我们还研究了hERG中C型失活的机制 K+通道，其在Shaker中与C型失活具有有趣的功能差异 通道，并且对于正常心脏功能是生理上关键的（目的3）。研究 提出的是重要的，因为它提供了更深入的了解电压的功能机制 Kv通道的门控和C型失活。这项研究也很重要，因为它将提供一个 使用UAA诱变研究主链H-键作用的一般策略， 离子结合位点，其对于许多膜蛋白家族的功能是重要的。