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。在中部 神经系统，它在秒的时间尺度上影响神经元的放电模式，并受损/改变 失活会导致各种神经疾病。神经元中传导的起始和持续时间 电压门控K+通道的状态，其由激活和失活的相互作用直接调制， 动作潜在射击率是其基础。在心脏中，HERG基因极快的C型失活 通道在心肌细胞的复极过程中起着重要作用。因此，理解分子基础 灭活的情况对人类健康有直接影响。PH激活的细菌KCSA通道是一种至关重要的 重要的原型模型系统；有强有力的证据表明，该通道的C型失活状态 是由选择性过滤器的收缩引起的。我们的主要假设是C型失活是一种 竞争因素控制的选择性滤膜的构象变化：局部堆积和氢 键合相互作用建立导电过滤器的固有热力学稳定性，同时还 稳定/不稳定是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。这个 这项拟议研究的目标是检验我们的假设并描绘出 K+通道的分子选择性过滤。这将通过依靠多学科的 将计算方法和实验方法相结合的策略。在目标1中，我们将研究分子 KCSA通道激活/失活变构耦合的决定因素。我们将生成一个 广泛的马尔可夫状态模型(MSM)包含打开细胞内的所有微观事件 栅极、离子传导和进入失活状态 无偏MD轨迹，以便提供激活/失活的完整计算范例 KCSA中的浇注工艺。我们将用工程振荡器样突变来确定KCSA的X射线结构 已知会影响C型失活，并用MD来表征这些系统。在目标2中，我们将调查 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 通道和钙激活的阳离子通道NaKTS通道。最后，在目标3中，我们将调查 电压门控K+通道Shaker、Kv1.2和Herg激活/失活的分子决定因素 使用X射线结晶学、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.