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: 9764015
• 负责人: Francis Valiyaveetil
• 金额: $ 35.15万
• 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/9764015
• 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k+（KV）通道将K+的通量与膜电势并播放钥匙 在大脑和心脏中的角色。 KV通道中的突变会导致人类严重疾病 例如癫痫和心律不齐。结构取得了重大进展 确定KV通道。尽管有可用的结构信息，但仍有主要 有关KV渠道中功能机制的问题，这些机制仍未得到答复。我们在这里 研究调节K+通量的电压门控和C型失活的过程 通过KV频道。我们使用以非自然氨基酸为中心的多学科方法 （UAA）在我们的研究中诱变。 UAA诱变是一种非常有力的方法 与传统诱变相比，蛋白质修饰，因为它允许大量 侧链修饰，还允许修饰蛋白质主链。我们使用这个 研究主链H键在第四跨膜螺旋中的作用的方法 （TM4）在振动器K+通道的电压门控和超极化激活和循环 核苷酸门控离子通道HCN（AIM 1）。我们研究了离子结合位点在 C型灭活中振动筛通道的选择性过滤器，我们补充功能 在KVAP通道上进行结构研究的振动摇具，这是一个古老的同源物 振动频道（AIM 2）。我们还研究了HERG中C型灭活的机制 k+通道，在振动器中与C型失活有有趣的功能差异 通道，对正常心脏功能至关重要（AIM 3）。研究 提议非常重要，因为它可以更深入地了解电压的功能机理 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诱变来研究主链H键和 离子结合位点，对于许多膜蛋白家族的功能很重要。