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 20 世纪 70 年代以来进行的膜片钳实验提供了打开和关闭的时间尺度 过去 20 年，X 射线晶体学的研究已经产生了离子的高分辨率结构。 迄今为止，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。 在这个提案中，我们通过利用新技术进步将动态与结构联系起来。 能够在离子通道上进行二维红外 (2D IR) 测量 2D 固有的时间分辨率。 红外光谱为几皮秒——比离子通道的毫秒运动快得多。 分辨率由主链羰基振动和静电荷之间的耦合产生，例如 残基特异性到域特异性的结构分辨率是通过常规同位素标记获得的。 Valiyaveetil 实验室开发的半合成程序现在可以很好地计算二维红外光谱。 准确地从短分子动力学轨迹，实现实验之间的一对一比较 和结构或提出的结构模型的二维红外、半合成和分子动力学的组合。 模拟为离子通道结构动力学提供了新的视角。在本提案中，我们讨论了两个问题。 在目标 1 和 2 中，我们研究了钾离子通道界的突出争议。 有争议的离子渗透通过 KcsA 和 NaK 选择性过滤器的新模型，称为“硬敲” 硬敲模型似乎可以解释 X 射线数据和所有其他现有的测量结果，甚至。 尽管它与教科书中最初的“敲击”模型根本不一致，但在目标 3 中，我们电压- 触发 KvAP 通道电压传感域 (VSD) 的结构运动，以研究 假设 VSD 中的重要 TM4 螺旋在 电压门控，如之前提出的，但从未通过直接结构或时间分辨建立 这些目标为离子通道中的突出问题提供了新的科学见解。 社区并建立研究通道结构动力学的新技术。