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

基本信息

批准号：
10241999
负责人：
Francis Valiyaveetil
金额：
$ 31.72万
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 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10241999
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

项目摘要

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 K+ (Kv) 通道将 K+ 通量与膜电位耦合并发挥关键作用在大脑和心脏中的作用。 Kv通道突变可导致人类严重疾病例如癫痫和心律失常。结构上有了重大进步 Kv 通道的测定。尽管有可用的结构信息，但仍存在主要的关于 Kv 通道的功能机制的问题仍未得到解答。在这里我们研究调节 K+ 通量的电压门控和 C 型失活过程通过Kv通道。我们采用以非天然氨基酸为中心的多学科方法（UAA）在我们的研究中发生诱变。 UAA诱变是一种非常有效的方法与传统的诱变相比，蛋白质修饰，因为它允许多种侧链修饰还允许修饰蛋白质主链。我们用这个研究第四跨膜螺旋中主链氢键作用的方法 (TM4) Shaker K+ 通道的电压门控以及超极化激活和循环核苷酸门控离子通道 HCN（目标 1）。我们研究了离子结合位点在 C 型失活中 Shaker 通道的选择性过滤器，我们补充了功能对 Shaker 的研究以及对 KvAP 通道的结构研究，KvAP 通道是摇床通道（目标 2）。我们还研究了 hERG 中 C 型失活的机制 K+ 通道，与摇床中的 C 型失活有有趣的功能差异通道，对于正常心脏功能具有重要的生理意义（目标 3）。研究提出的建议很重要，因为它提供了对电压功能机制的更深入的了解 Kv 通道中的门控和 C 型失活。这项研究也很重要，因为它将提供使用 UAA 诱变研究主链氢键作用的一般策略和离子结合位点，对于许多膜蛋白家族的功能很重要。