Computational Studies of Ion Channels

K+通道的C型失活是一个具有重要生理意义的分子过程。中部 神经系统，它会影响神经元的放电模式在秒的时间尺度上，并受损/改变 失活导致多种神经障碍。在神经元中， 电压门控K+通道的状态，其通过激活和失活的相互作用直接调节， 是动作电位放电率的基础在心脏中，hERG的极快C型失活 通道在心肌细胞复极化过程中起重要作用。因此，了解分子基础 失活对人体健康有直接影响。pH激活的细菌KcsA通道是一个关键的 重要的原型模型系统;有强有力的证据表明，C型失活状态的这一渠道 是由选择性过滤器的收缩引起的。我们的总体假设是，C型失活是一个 选择性过滤器的构象变化受竞争因素控制：局部堆积和氢 键合相互作用建立了导电过滤器的固有热力学稳定性，同时还 稳定化/去稳定化通过与细胞内门的变构偶联或通过- 在电压激活通道的情况下，孔域与电压传感器的空间相互作用。的 这项研究的目的是验证我们的假设，并描绘构象可塑性的 K+通道的选择性过滤。这将通过依靠多学科 结合计算和实验方法的策略。在aim 1中，我们将研究 KcsA通道中激活/失活变构偶联的决定因素。我们将生成一个 广泛的马尔可夫状态模型（MSM），包括所有的微观事件，打开细胞内的 门，离子传导，并进入失活的基础上，从大量的聚合数据， 无偏MD轨迹，以便提供激活/失活的完整计算范例 KcsA中的门控过程。我们将确定具有工程化Shaker样突变的KcsA的X射线结构 已知影响C型失活，并用MD表征这些系统。在目标2中，我们将研究 由阳离子NaK构建的嵌合细菌通道中选择性过滤器的结构多态性 通道和钙激活阳离子通道NaKTs通道。最后，在目标3中，我们将研究 电压门控K+通道Shaker、Kv1.2和hERG中激活/失活的分子决定因素 使用X射线晶体学、功能测量和MD模拟。

C-type inactivation of K+ channels is a molecular process of great physiological significance. In the central nervous system, it affects the firing patterns of neurons on the time-scale of seconds, and impaired/altered inactivation leads to a variety of neurological disorders. In neurons the onset and duration of the conductive state of voltage-gated K+ channels, which are directly modulated by the interplay of activation and inactivation, underlie the action-potential firing rates. In the heart, the extremely fast C-type inactivation of the hERG channel plays an important role in the repolarization of cardiac cells. Thus, understanding the molecular basis of inactivation has a direct impact on human health. The pH-activated bacterial KcsA channel is a critically important prototypical model system; there is strong evidence that the C-type inactivated state of this channel is caused by a constriction of the selectivity filter. Our overarching hypothesis is that C-type inactivation is a conformational change of the selectivity filter controlled by competing factors: local packing and hydrogen bonding interactions establish the inherent thermodynamic stability of the conductive filter, while further stabilization/destabilization is communicated via the allosteric coupling with the intracellular gate, or through- space interactions of the pore domain with the voltage sensor in the case of voltage-activated channels. The goal of the proposed research is to test our hypothesis and delineate the conformational plasticity of the selectivity filter of K+ channels in molecular terms. This will be achieved by relying on a multidisciplinary strategy that combines computational and experimental approaches. In aim 1, we will study the molecular determinants of the activation/inactivation allosteric coupling in the KcsA channel. We will generate an extensive Markov State Model (MSM) encompassing all the microscopic events of opening the intracellular gate, ion conduction, and entry into inactivation on the basis of aggregate data from a large number of unbiased MD trajectories in order to provide a complete computational paradigm of the activation/inactivation gating process in KcsA. We will determine the X-ray structure of KcsA with engineered Shaker-like mutations known to affect C-type inactivation and characterize these systems with MD. In aim 2, we will investigate the structural polymorphism of the selectivity filter in chimeric bacterial channels built from the cationic NaK channel and the calcium-activated cationic channel NaKTs channel. Finally, in aim 3, we will investigate the molecular determinants of activation/inactivation in the voltage-gated K+ channels Shaker, Kv1.2 and hERG using X-ray crystallography, functional measurements, and MD simulations.