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

使用 2D IR 对离子电导和离子通道结构动力学的新视角

• 批准号: 10405536
• 负责人: Francis Valiyaveetil
• 金额: $ 34.1万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10405536
• 关键词: Address; Arrhythmia; Binding Sites; Biological; Cell membrane; Charge; Collaborations; Communities; Data; Dependence; Development; Disease; Electrostatics; Epilepsy; Family; Frequencies; Human; Integral Membrane Protein; Ion Channel; Ion Transport; Ions; Isotope Labeling; Kinetics; Lead; Light; Measurement; Measures; Membrane; Membrane Proteins; Methodology; Modeling; Molecular Conformation; Motion; Mutation; Physiology; Play; Potassium Channel; Procedures; Proteins; Publications; Research; Resolution; Roentgen Rays; Role; Science; Series; Site; Spectrum Analysis; Structural Models; Structure; Techniques; Technology; Testing; Textbooks; Time; Variant; Vertebral column; Water; Work; X-Ray Crystallography; absorption; base; electric field; experimental study; fascinate; innovation; insight; ion dynamics; millisecond; molecular dynamics; patch clamp; two-dimensional; vibration; voltage

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.

抽象的 自1970年代以来进行的斑块夹实验提供了开放和关闭的时间尺度 离子通道。在过去的20年中，X射线晶体学产生了离子的高分辨率结构 频道。到目前为止，还没有关于通道动力学或施加电压对离子的影响的直接实验 通道结构。在此提案中，我们通过利用新的技术进步将动态连接到结构 这使离子通道上的二维红外（2D IR）测量。 2D的继承时间分辨率 红外光谱是几次二秒 - 比离子通道的毫秒动作快得多。结构 分辨率来自骨干羰基振动与静电电荷之间的耦合 离子。通过同位素标记，可以通过同位素标记进行常规，以特定于域特异性的结构分辨率。 在Valiyaveetil实验室开发的半合成程序。而且，现在可以非常计算2D红外光谱 准确地从短分子动力学轨迹中准确，实现了实验之间的一对一比较 以及结构或建议的结构模型。 2D IR，半合成和分子动力学的组合 模拟允许对离子通道结构动力学有了新的视角。在此提案中，我们解决了两个 钾离子频道社区中的杰出争议。在目标1和2中，我们调查 通过KCSA和NAK的选择性滤波器，有争议的离子渗透模型，称为“硬敲击” 模型。硬敲击模型似乎可以解释X射线数据和所有其他现有测量结果，甚至 尽管从根本上讲，它与教科书中的原始“敲门”模型不一致。在AIM 3中，我们电压 - 触发KVAP通道的电压传感域（VSD）的结构运动，以研究 VSD中必需的TM4螺旋在vSD中经历构象和/或水分变化的假设 如前所述，但从未建立直接结构或时间分辨的电压门控 测量。这些目的为离子频道中的杰出问题提供了新的科学见解 社区并建立一种用于研究离子通道结构动力学的新技术。