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+通道是膜蛋白，可促进K+离子跨细胞膜的转运。他们在神经和肌肉放松，认知和血压调节中起关键作用。尽管多年来对K+通道进行了广泛的研究，但离子通道激活的机制仍然是一个正在进行的辩论的话题，这种辩论是无法通过已建立的实验方法解决的。二维红外（2D IR）光谱是一种新兴的分析技术，它以化学键特异性空间和高时间分辨率探测蛋白质的结构变化。最新的技术进步允许在生理上现实的条件下将2D红外光谱法应用于离子通道。该项目将分子动力学模拟与计算光谱法结合了研究K+通道的激活机制。模拟将通过2D IR实验进行验证，进而将用于设计新的实验，以提供对K+通道激活机制的最清晰洞察力。提出的工作将得出K+通道功能的原子级描述。了解功能的K+通道的基础机制是确定严重疾病（例如心律不齐和癫痫病）的关键因素。它提供了设计用于离子通道药理学疗法的前景。该研究基础设施改进Track-4 Epscor Research Fellows（RII Track-4）项目将为特拉华大学（UD）的助理教授提供奖学金。电压门控的K+通道（KV）是积分的膜蛋白，根据电化学梯度有选择性地在细胞膜上进行K+离子。 KV的电压感应结构域（VSD）感觉在膜电势中发生变化，并触发构象变化，从而导致通道的打开和关闭。尽管有大量的NMR和X射线晶体学数据，但有关VSD功能的许多基本问题仍然开放。已经假设VSD的S4螺旋段在电压门控过程中经历了构象和/或水分变化，但是由于大多数生物物理物理技术技术无法在施加电压下执行，因此从未通过直接的结构测量来检验该假设。 2D红外光谱可以探测具有特定位点特异性分辨率的蛋白质结构，并且在包括施加电压在内的生理条件下。它还可以探测在毫秒时尺度上发生的VSD的门控动力学。最近，2D红外光谱法的灵敏度已显着提高，使其独特地适合研究离子通道。所提出的工作将将分子动力学模拟与计算2D IR光谱法相结合，以阐明电压激活过程中VSD的构象和水分变化。 VSD的现有结构模型将对2D IR实验进行模拟和测试。基于与实验的比较，将改进模型，否则将开发新的模型。模拟还将用于设计新的2D IR实验。我们旨在确定VSD的相关构象及其在电压激活期间发生的顺序。该奖项反映了NSF的法定任务，并被认为是值得通过基金会的知识分子优点和更广泛的影响审查标准通过评估来获得支持的。