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

蛋白质-蛋白质和蛋白质-H2O氢键与选择性过滤动力学有关 和整体功能。在该联盟的第二阶段，我们将继续研究这些事件，结合 光谱学、结晶学、电生理学和计算方法来探索 K+选择滤光片的不同选通方式。除了我们的基本实验模型(KCSA)之外，我们现在还有 添加了第二个系统，在总体构象方面具有显著的选择性过滤可塑性。这个 NAK-HERG结构经历了戏剧性的结构重新安排，以响应两种含义的变化 离子和临床相关突变的结果(与长QT综合征有关)。正因为如此 发现，我们能够在原子水平上分析更广泛的构象动力学 通过多个时间尺度进行选择性过滤。这一独特的结构动力学数据集将使我们能够 详细的计算分析，以提供连接两者的能源格局的明确描述 静态结构及其构象途径和相关的功能结果。 我们提出了一种使用无细胞和半合成KCSA和NAK-HERG，膜片钳，X-HERG的协同方法 射线衍射和最新的光谱方法(核磁共振和单核磁共振)在切割的辅助下发展起来 边缘计算方法，对选择性发生的构象变化的理解 在停用时和离子传导过程中过滤。这些总体目标将通过以下方式实现 具体目标： 1.确定与离子相互作用有关的分子运动、总能量和作用 K+选择滤膜在KCSA和NAK-Herg嵌合体中的构象灵活性。 2.建立了选择性滤池结构水动力学对KCSA C型失活动力学的影响 以及连接所有关键浇口中间件的充满活力的规则，这些规则使用试验性和 计算方法。 我们计划解决的问题类型只能是因为健壮性和 现有系统的实验成熟度(KCSA和NAK-HERG)。对中国能源前景的估计 构象的宇宙和高带宽电生理学将使我们能够建立 描述与K+相关联的广泛运动、动力学和门控模式的一般规则 选择性过滤器。这些数据预计将提供对选择性过滤门控机制的确切理解 这两个模型系统，预计也将适用于K+通道超家族的其他成员。

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.