Measuring ion channel pore dynamics with fluorescence

描述(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)，包括动作电位复极和节律控制 射击模式。控制功能结果的通道属性 通道活跃度包括电流大小和通道门控速率 事件(激活、停用和停用)。功能丰富的外墙 钾通道孔的前庭/选择性过滤器区域已被 被认为在开放状态下只有一种构象，并且具有 对开放通道功能的调制作用很小或没有作用。最近，我们 描述了一种新的机制，通过它，电流大小，激活率和 失活率，以及内外经络药理， 受外源钾浓度的相关变化的调节。我们 演示了这些频道属性的更改，这些属性可以 大量的，由一种以前未知的构象变化引起的 它发生在毛孔的外部前庭。此外，这一点 只有在以下情况下才能观察到外前庭的构象变化 与结构灵活的选择性过滤器一致的显示属性 毛孔的区域。这些结果表明，与之相反， 之前人们认为，一些离子通道中的渗透途径 有很大程度的“结构灵活性”，而这 “灵活性”可以显著影响开放渠道的功能。也不是本质上的 构象变化，也没有详细了解它是如何被调控的， 仅通过电生理技术即可获得。这样做的目的是 应用是集成两种复杂的荧光技术， 荧光猝灭和荧光共振能量转移(FRET)， 我们实验室的膜片钳电生理学。这将使我们能够直接 检查构象变化的性质，以及 控制构象变化。它还将允许我们测试 一种全新的假说，即在结构灵活性方面的差异 渗透途径在一定程度上是功能调节差异的基础。 密切相关的离子通道。整合这项技术的能力 我们的研究将为我们的离子研究提供一种新的和增强的方法 渠道机制，并将使我们能够收集必要的初步数据 随后的资金。

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.