Structural Basis of Coupling and Dynamics in K+ Channels

项目摘要 作为对膜电位去极化的响应，电压依赖通道经历了一系列 从非导电状态(闭合)到活化(导电)的构象变化，最终 稳定在不导通的失活状态。K+通道功能已经与这样的基础相关联 细胞起着调节电活动、信号转导和渗透平衡的作用。在更高的 生物体，K+通道功能障碍可能会导致不受控制的电过度兴奋时期，如 癫痫发作、肌强直和心律失常。因此，了解K+通道的努力 结构、功能和动力学直接关系到人类的健康和疾病。持续的长期 本项目的目标是进一步了解电压依赖门控的分子机制。 通过重点分析原核和真核系统中的K+通道门控。 具体来说，我们将解决以下关键问题：键的原子结构是什么 决定通道活动的构象？这个问题的答案将针对嵌入的薄膜 系统以及在格子中排序的系统。电压依赖性的分子基础是什么 门禁？我们将测试这样的假设，即特定的滑动螺旋运动(单击运动)可以 解释某些电压传感器中的电荷转移，但可能不解释其他传感器。传感器充电越多 移位，其传感器需要移动的点击次数就越多。以及渠道的不同部分是如何 互动以定义开放渠道活动？我们计划结合光谱学来研究这些问题 技术(电子顺磁共振和核磁共振)、X射线结晶学、电生理和计算方法。我们 打算通过研究大量生物化学定义的物质来继续这些结构-功能研究 从KCSA和KvAP到振动器电压传感器和超极化激活通道的系统 来自简氏甲烷球菌(MVP)。此外，我们还将重点关注电压传感技术。 Ci-VSP的结构域，旨在改善 我们最近的晶体结构的分辨率。最后，我们将研究人类电压的结构- 膜上依赖的质子通道Hv1通过广泛的定点自旋标记分析和 计算建模。这项提议应该开辟新的实验途径，将有助于我们的 了解重要的生物事件，如电信号、信号转导和离子 频道选通。

Project Summary In response to membrane potential depolarization, voltage-dependent channels undergo a series of conformational changes from a non-conducting state (closed) to an activated (conducting), finally stabilizing in a non-conducting inactivated state. K+ channel function has been associated with such basic cellular functions as the regulation of electrical activity, signal transduction and osmotic balance. In higher organisms, K+ channel dysfunction may lead to uncontrolled periods of electrical hyperexcytability, like epileptic episodes, myotonia and cardiac arrhythmia. Consequently, efforts to understand K+ channel structure function and dynamics relate directly to human health and disease. The continuing long-term goal of this project is to further understand the molecular mechanisms of gating in voltage-dependent channels, by focusing on the analysis of K+ channel gating in prokaryotic and eukaryotic systems. Specifically we will address the following key questions: What are the atomic structures of the key conformations that determine channel activity? This question will be answered for membrane embedded systems as well as those ordered in a lattice. What are the molecular bases of voltage-dependent gating? We will be testing the hypothesis that a specific sliding helix movement (the one click motion) can explain charge translocation in certain voltage sensors, but perhaps not others. The more charge a sensor translocates, the larger the number of clicks its sensor needs to move. And how different parts of the channel interact to define open channel activity? We plan to study these problems by combining spectroscopic techniques (EPR and NMR), X-ray crystallography electrophysiological and computational methods. We intend to continue these structure-function studies by investigating a wealth of biochemically-defined systems from KcsA and KvAP, to the Shaker voltage sensor and the hyperpolarization-activated channel from Methanococcus janschii (MVP). In addition, we will focus our attention on the voltage-sensing domain from the Ciona intestinalis-Voltage-Sensor-containing Phosphatase (Ci- VSP) aiming to improve the resolution of our recent crystals structures. Finally, we will examine the structure of the human voltage- dependent proton channel Hv1 in membranes though an extensive site-directed spin labeling analysis and computational modeling. This proposal should open new experimental avenues that will contribute to our understanding of biologically important events such as electrical signaling, signal transduction and ion channel gating.