A new perspesective on ion conductance and structural dynamics of ion channels using 2D IR

摘要 自20世纪70年代以来进行的膜片钳实验提供了开放和关闭的时间尺度 离子通道。在过去的二十年里，X射线结晶学已经产生了离子的高分辨率结构。 频道。到目前为止，还没有关于离子通道动力学或施加电压对离子的影响的直接实验。 航道结构。在这项提议中，我们通过利用新的技术进步将动力与结构联系起来 这使得在离子通道上进行二维红外(2D IR)测量成为可能。2D的固有时间分辨率 红外光谱只有几皮秒，比离子通道的毫秒运动快得多。结构性 解析度源于主链羰基振动和静电电荷之间的耦合，例如 离子。通过常规的同位素标记获得特定于残基到特定结构域的结构解析 Valiyaveetil实验室开发的半合成程序。而且，2D红外光谱现在可以计算出 准确地从短的分子动力学轨迹，使一对一的实验之间的比较 和结构或建议的结构模型。这种2D IR、半合成和分子动力学的结合 模拟为离子通道结构动力学提供了一个新的视角。在这项提案中，我们涉及两个问题 钾离子通道领域的突出争议。在目标1和目标2中，我们调查了 有争议的离子通过KCSA和NaK选择性过滤器的新模型，称为“硬敲击” 模特。硬敲击模型似乎解释了X射线数据和所有其他现有的测量，甚至 尽管它从根本上与教科书中最初的“连锁反应”模式不同。在目标3中，我们给- 触发KvAP通道电压感应域(VSD)的结构运动，以研究 假设室间隔缺损中重要的TM4螺旋经历构象和/或水化变化 电压门控，如先前提出的，但从未由直接结构或时间分辨建立 测量。这些目标为离子通道中的突出问题提供了新的科学见解 并建立了一种研究离子通道结构动力学的新技术。

Abstract Patch clamp experiments performed since the 1970s have provided the timescales for the opening and closing of ion channels. X-ray crystallography over the past 2 decades has yielded high-resolution structures of ion channels. As yet, there are no direct experiments on channel dynamics or the effect of an applied voltage on ion channel structures. In this proposal, we connect dynamics to structure by leveraging new technological advances that enable two-dimensional infrared (2D IR) measurements on ion channels. The inherent time-resolution of 2D IR spectroscopy is a few picoseconds – much faster than the millisecond motions of ion channels. Structural resolution arises from couplings between the backbone carbonyl vibrations and electrostatic charges such as ions. Residue-specific to domain-specific structural resolution is obtained with isotope labeling made routine by semisynthesis procedures developed in the Valiyaveetil lab. And, 2D IR spectra can now be calculated very accurately from short molecular dynamics trajectories, enabling a one-to-one comparison between experiment and structure or proposed structural models. This combination of 2D IR, semisynthesis, and molecular dynamics simulations permit a new perspective on ion channel structural dynamics. In this proposal, we address two outstanding controversies in the potassium ion channel community. In Aims 1 and 2, we investigate a controversial new model for ion permeation through the selectivity filter of KcsA and NaK, called the “hard-knock” model. The hard-knock model appears to explain the X-ray data and all other existing measurements, even though it is fundamentally at odds with the original “knock-on” model found in textbooks. In Aim 3, we voltage- trigger the structural motions of the voltage sensing domain (VSD) of the KvAP channel, to investigate the hypothesis that the essential TM4 helix in the VSD undergoes a conformational and/or hydrational change during voltage gating, as previously proposed but never established by a direct structural or time-resolved measurement. These aims provide new scientific insights into outstanding problems in the ion channel community and establish a new technique for studying ion channel structural dynamics.