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

K+通道是促进K+离子跨细胞膜运输的膜蛋白。它们在神经和肌肉放松、认知和血压调节方面起着关键作用。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，用现有的实验方法是不可能解决的。二维红外光谱(2D IR)是一种新兴的分析技术，它以特定的化学键、特定的空间和高的时间分辨率探测蛋白质的结构变化。最近的技术进步允许将2D IR光谱应用于生理上真实条件下的离子通道。本项目将分子动力学模拟与计算光谱学相结合，研究K+通道的激活机制。模拟将通过2D IR实验进行验证，并将被用于设计新的实验，这些实验可以为K+通道的激活机制提供最清晰的见解。这项工作将推导出K+通道功能的原子级描述。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的原因的关键因素。它为设计离子通道药理学的治疗方法提供了前景。研究基础设施改进Track-4 EPSCoR研究人员(RII Track-4)项目将为特拉华大学(UD)的一名助理教授提供研究金。电压门控K+通道(Kv)是一种完整的膜蛋白，它根据电化学梯度选择性地将K+离子输送到细胞膜上。Kv的电压敏感结构域(VSD)感测膜电势的变化，并触发构象变化，导致通道的打开和关闭。尽管有丰富的核磁共振和X射线结晶学数据，但许多关于VSD功能的基本问题仍然悬而未决。人们假设VSD的S4螺旋段在电压门控过程中经历了构象和/或水化变化，但这一假设从未被直接结构测量验证，因为大多数生物物理结构技术不能在施加电压下进行。二维红外光谱可以在包括外加电压在内的生理条件下，以特定位置的分辨率探测蛋白质结构。它还可以探测发生在毫秒时间尺度上的VSD的门控动态。近年来，二维红外光谱的灵敏度显著提高，特别适合于离子通道的研究。这项工作将结合分子动力学模拟和计算二维红外光谱来阐明电压激活过程中VSD的构象和水化变化。现有的VSD结构模型将根据2D IR实验进行模拟和测试。在与实验对比的基础上，对模型进行改进，或开发新的模型。还将利用模拟来设计新的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的出现顺序。这一裁决反映了NSF的法定使命，并已通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.