Measuring ion channel pore dynamics with fluorescence

用荧光测量离子通道孔隙动力学

• 批准号: 6414251
• 负责人: Stephen J Korn
• 金额: $ 15.15万
• 依托单位: UNIVERSITY OF CONNECTICUT STORRS
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-01 至 2003-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6414251
• 关键词: aminoacid; biological transport; cell line; conformation; electrophysiology; electroporation; fluorescence; fluorescence resonance energy transfer; fluorescent dye /probe; human genetic material tag; ion transport; membrane activity; membrane permeability; membrane potentials; membrane structure; potassium channel; potassium ion; sodium ion; technology /technique development; tissue /cell culture; voltage /patch clamp

DESCRIPTION (provided by applicant): Voltage-activated potassium channels serve several critical functions in all excitable cells (brain, heart, endocrine and muscle), including repolarization of action potentials and control of rhythmic firing patterns. Channel properties that control the functional outcome of channel activity include current magnitude and the rate of channel gating events (activation, inactivation and deactivation). The functionally rich outer vestibule/selectivity filter region of the potassium channel pore has been considered to have just a single conformation in the open state, and have little or no role in the modulation of open channel function. Recently, we described a novel mechanism by which current magnitude, activation rate and inactivation rate, as well as both internal and external channel pharmacology, are modulated by relevant changes in external potassium concentration. We demonstrated that changes in these channel properties, which can be substantial, result from a previously unknown type of conformational change that occurs in the outer vestibule of the pore. Furthermore, this conformational change in the outer vestibule is observed only in channels that display properties consistent with a "structurally flexible" selectivity filter region of the pore. These results suggest the possibility that, in contrast to what has been previously believed, the permeation pathway in some ion channels has a significant degree of "structural flexibility," and that this "flexibility" can markedly affect open channel function. Neither the nature of the conformational change, nor a detailed understanding of how it is regulated, can be obtained solely with electrophysiological techniques. The goal of this application is to integrate two sophisticated fluorescence techniques, fluorescence quenching and fluorescence resonance energy transfer (FRET), with patch clamp electrophysiology in our lab. This will allow us to directly examine the nature of the conformational change, and the mechanisms that control the conformational change. It will also allow us to test the fundamentally novel hypothesis that differences in "structural flexibility" of the permeation pathway underlie, in part, differences in functional regulation of closely related ion channels. The ability to incorporate this technology into our research will provide a new and enhanced approach for our study of ion channel mechanisms, and will allow us to collect preliminary data necessary for subsequent funding.

描述（由申请人提供）：电压激活钾通道 所有可兴奋细胞（脑、心脏、内分泌和 肌肉），包括动作电位的复极和节律性 发射模式。控制的功能结果的通道属性 通道活动包括电流大小和通道门控速率 事件（激活、停用和停用）。功能丰富的外层 钾通道孔的前庭/选择性过滤区已经被 被认为在开放状态下只有单一构象，并且具有 在开放通道功能的调节中作用很小或没有作用。最近我们 描述了一种新的机制，通过该机制，电流大小、激活率和 失活率，以及内部和外部通道药理学， 受外部钾浓度相关变化的调节。我们 证明了这些通道特性的变化， 大量，由以前未知类型构象变化引起 发生在毛孔的外前庭。而且这 仅在通道中观察到外前庭的构象变化， 显示与“结构灵活”的选择性过滤器一致的特性 孔的区域。这些结果表明，与 以前认为，某些离子通道中的渗透途径 具有很大程度的“结构灵活性”， “柔性”对明渠功能影响显著。无论是自然的 构象的变化，也没有详细了解它是如何调节的， 可以单独通过电生理技术获得。这个目标 应用是整合两种复杂的荧光技术， 荧光猝灭和荧光共振能量转移（FRET）， 膜片钳电生理学。这将使我们能够直接 研究构象变化的性质，以及 控制构象变化。这也将使我们能够测试 一个全新的假设，即在“结构灵活性”的差异， 渗透途径部分是功能调节差异的基础 与之密切相关的离子通道将这项技术 将为我们的离子研究提供一种新的、更好的方法 渠道机制，并使我们能够收集必要的初步数据， 后续融资。