RII Track-4:NSF: Unravelling Gating Mechanisms of Ion Channels Using Computational and Experimental Ultrafast Vibrational Spectroscopy

RII Track-4：NSF：利用计算和实验超快振动光谱揭示离子通道的门控机制

• 批准号: 2229651
• 负责人: Alexei Kananenka
• 金额: $ 19.36万
• 依托单位: University of Delaware
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2229651&HistoricalAwards=false
• 关键词: RII; Track; NSF; Unravelling; Gating

K+ channels are membrane proteins that facilitate the transport of K+ ions across the cell membranes. They play key roles in nerve and muscle relaxation, cognition, and regulation of blood pressure. While K+ channels have been extensively studied over many years, the mechanism of activation of ion channels remains a topic of ongoing debate that has not been possible to solve with established experimental methods. Two-dimensional infrared (2D IR) spectroscopy is an emerging analytical technique that probes structural changes of proteins with chemical bond specific spatial and high temporal resolution. Recent technological advances permit applications of 2D IR spectroscopy to ion channels under physiologically realistic conditions. This project combines molecular dynamics simulations with computational spectroscopy to study activation mechanisms of K+ channels. Simulations will be validated by 2D IR experiments and, in turn, will be used to design new experiments that can provide most clear insight into activation mechanisms of K+ channels. The proposed work will derive the atomistic-level description of function of K+ channels. Understanding the mechanisms underlying K+ channels functioning is a key factor in determining the cause of severe diseases such as cardiac arrhythmias and epilepsies. It offers the prospect of designing therapies for ion channel pharmacology. This Research Infrastructure Improvement Track-4 EPSCoR Research Fellows (RII Track-4) project would provide a fellowship to an Assistant Professor at the University of Delaware (UD). Voltage-gated K+ channels (Kv) are integral membrane proteins that selectively conduct K+ ions across cell membranes according to the electrochemical gradient. The voltage-sensing domain (VSD) of Kv senses changes in the membrane electrical potential and triggers a conformational change resulting in the opening and closing of the channel. Despite a wealth of NMR and X-ray crystallography data, many fundamental questions regarding the function of the VSD remain open. It has been hypothesized that the S4 helical segment of the VSD undergoes a conformational and/or hydrational change during voltage-gating, but this hypothesis has never been tested by a direct structural measurement because most biophysical structural techniques cannot be performed under applied voltage. 2D IR spectroscopy can probe protein structures with site-specific resolution and under physiological conditions including applied voltage. It can also probe the gating dynamics of the VSD occurring on the millisecond timescale. Recently the sensitivity of 2D IR spectroscopy has been significantly increased, making it uniquely suitable to study ion channels. The proposed work will combine molecular dynamics simulations with computational 2D IR spectroscopy to elucidate the conformational and hydrational changes of the VSD during voltage activation. The existing structural models of the VSD will be simulated and tested against 2D IR experiments. Based on the comparison with experiments, the models will be refined, or new models will be developed. Simulations will also be utilized to design new 2D IR experiments. We aim to determine the relevant conformations of the VSD and the order they occur during voltage activation.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

K+通道是促进K+离子跨细胞膜转运的膜蛋白。它们在神经和肌肉放松、认知和血压调节中起着关键作用。虽然K+通道已被广泛研究多年，但离子通道的激活机制仍然是一个持续争论的话题，无法用已建立的实验方法解决。二维红外光谱技术是一种新兴的分析技术，它以高的时间分辨率和空间分辨率探测蛋白质的结构变化。最近的技术进步允许在生理现实条件下将2D IR光谱应用于离子通道。本计画结合分子动力学模拟与计算光谱学来研究钾离子通道的活化机制。模拟将通过二维红外实验进行验证，反过来，将用于设计新的实验，可以提供最清晰的洞察K+通道的激活机制。本文的工作将从原子水平上对K+通道的功能进行描述。了解K+通道功能的机制是确定严重疾病（如心律失常和癫痫）原因的关键因素。为离子通道药理学的治疗设计提供了前景。这个研究基础设施改善轨道-4 EPSCoR研究员（RII轨道-4）项目将提供奖学金，以助理教授在特拉华州（UD）的大学。电压门控K+通道（Kv）是根据电化学梯度选择性地将K+离子传导穿过细胞膜的膜蛋白。Kv的电压敏感域（VSD）感知膜电位的变化并触发构象变化，从而导致通道的打开和关闭。尽管有丰富的核磁共振和X射线晶体学数据，许多关于VSD功能的基本问题仍然是开放的。有人假设，室间隔缺损的S4螺旋段在电压门控期间经历构象和/或水合变化，但这一假设从未通过直接结构测量进行过测试，因为大多数生物物理结构技术不能在施加电压下进行。二维红外光谱可以探测蛋白质结构与位点特异性的分辨率和生理条件下，包括施加电压。它还可以探测在毫秒时间尺度上发生的VSD的门控动力学。最近，二维红外光谱的灵敏度已显着增加，使其特别适合研究离子通道。拟议的工作将结合联合收割机分子动力学模拟与计算二维红外光谱，阐明电压激活过程中的VSD的构象和水化的变化。VSD的现有结构模型将被模拟和测试对2D IR实验。在与实验比较的基础上，将改进模型，或开发新的模型。模拟也将用于设计新的2D IR实验。我们的目标是确定VSD的相关构象以及它们在电压激活过程中发生的顺序。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。