Bridge 4: Dynamics of Ion Permeation and Conformational Coupling in K+channels

蛋白质-蛋白质和蛋白质-H2O氢键已被牵连在定义选择性过滤动力学 整体功能。在这个联合体的第二阶段，我们将继续研究这些事件， 光谱学、晶体学、电生理学和计算方法来探测 K+选择性滤波器中的不同选通模式。除了我们的基本实验模型（KcsA），我们现在有 增加了第二个系统，在整体构象方面具有显着的选择性过滤器可塑性。的 NaK-hERG构建体响应于渗透性细胞中的两种变化而经历显著的结构重排。 离子和临床相关突变的结果（与长QT综合征相关）。因为这些 研究结果，我们能够在原子水平上分析更广泛的构象动力学宇宙， 通过多个时间尺度进行选择性过滤。这个独特的结构动力学数据集将使我们能够 详细的计算分析，以提供一个明确的描述能源景观，连接每个 静态结构及其构象途径和相关功能后果。 我们提出了一种协调一致的方法，使用无细胞和半合成的KcsA和NaK-hERG，膜片钳，X-射线光电子显微镜， 射线衍射和最先进的光谱方法（NMR和ssNMR）的发展，与援助的切割 边缘计算方法，了解构象变化发生的选择性 过滤器时，它失活和离子传导。这些总体目标将通过以下方式实现： 具体目标： 1.确定分子运动，整体能量学和离子相互作用的作用， KcsA和NaK-hERG嵌合体中K+选择性过滤器的构象灵活性。 2.确定选择性过滤器结构水动力学对KcsA中C型失活动力学的作用 和充满活力的规则，连接所有关键门控中间体使用的实验和 计算方法。 我们计划解决的问题类型只能是因为鲁棒性和 现有系统（KcsA和NaK-hERG）的实验成熟度。2010 - 2011年能源状况估计 构象的宇宙加上高带宽的电生理学将使我们能够建立 描述与K+相关的广泛运动、动力学和门控模式的一般规则 选择性过滤器预计这些数据将提供一个坚定的理解选择性过滤器门控机制， 这两个模型系统，预计也适用于其他成员的K+通道超家族。

Protein-protein and protein-H2O hydrogen bonds have been implicated in defining selectivity filter dynamics and overall function. In Phase II of this consortium, we will continue to study these events by combining spectroscopic, crystallographic, electrophysiological and computational methods to probe the dynamics of the different gating modes in K+ selective filters. In addition to our basic experimental model (KcsA) we have now have added a second system with remarkable selectivity filter plasticity in regards to overall conformation. The NaK-hERG construct undergoes dramatic structural rearrangements in response to both changes in permeant ions and as a result of clinically relevant mutations (associated with the long QT syndrome). Because of these findings, we are in a position to analyze, at atomic level, a wider universe of conformational dynamics at the selectivity filter through multiple time scales. This unique structural dynamics dataset will allow us to pursue detailed computational analyses to provide a definitive description of the energy landscape that connects each static structure with its conformational pathway and associated functional consequences. We propose a concerted approach using cell free and semi-synthesis of KcsA and NaK-hERG, patch clamp, X- ray diffraction and state of the art spectroscopic methods (NMR and ssNMR) to develop, with the aid of cutting edge computational methods, an understanding of the conformational changes taking place at the selectivity filter when it inactivates and during ion conduction. These overall goals will be carried out through the following specific aims: 1. Determine the molecular motions, overall energetics and role of ion interactions associated with the conformational flexibility of K+ selective filter in KcsA and a NaK-hERG chimera. 2. Establish the role of selectivity filter structural water dynamics on the kinetics of C-type inactivation in KcsA and the energetic rules that connect all key gating intermediates using a combination of experimental and computational approaches. The types of questions we plan to address can be approached only because of the robustness and experimental maturity of the systems at hand (KcsA and NaK-hERG). Estimation of the energy landscape for the universe of conformations together with high bandwidth electrophysiology will allow us to establish the general rules that describe a wide range of motion, dynamics and gating modes associated with the K+ selectivity filter. This data is expected to provide a firm understanding of selectivity filter gating mechanisms in the two model systems and is expected to also apply to other members of the K+ channel superfamily.