K+ channel structural dynamics landscape: from selectivity to gating

钾离子通道结构动力学景观：从选择性到门控 使生命成为可能的化学和电子活动由一系列离子通道控制。这些跨膜蛋白调节细胞膜上离子的不同浓度，在K+通道的情况下，具有区分极其相似的离子(如Na+和K+)的非凡能力。虽然离子选择性和门控机制已经研究了很多年，但潜在的结构和时间动力学仍然知之甚少。使用诸如单分子FRET(SmFRET)或荧光非规范氨基酸掺入等技术，可以实时评估离子通道分子内的运动和状态。在这个项目中，我打算结合广泛的技术，从电生理学到单分子方法来描述合成和活细胞膜中K+通道的实时动态。通过结合我以前在离子通道生物物理学和电生理学方面的经验，以及建立新的方法和相关技术的培训，包括smFRET、高光谱成像、结构确定和分子动力学(MD)模拟，我提出了两个具体的目标来解决这个统一的问题：K+通道中离子选择性和门控的结构动力学是什么？这些研究将首先以NaK/NaK2K(非选择性和K+选择性)通道为模型，验证序列为TxGYG的钾通道选择性过滤器(SF)在没有K+的情况下变得动态和不稳定的假设，在K+的存在下获得更稳定的构象。这一目标将在K99阶段完成，为离子通道选择性提供新的见解。其他技术和理论技能的发展将使我能够扩大我在R00阶段的努力，以研究更复杂的通道中通道门控机制的结构动力学，包括与药物相关的真核双孔结构域K+通道家族(K2P)，它有助于兴奋和非兴奋组织中的泄漏电流。这个K+通道家族在许多生理和病理条件下起着关键作用，了解门控和选择性机制将有助于调节它们的行为，以克服不同的病理。总体而言，拟议的综合研究和培训将有助于我成为一名独立研究员，使用广泛的尖端技术来帮助更好地了解健康和疾病中的膜蛋白结构动力学。

K+ channel structural dynamics landscape: from selectivity to gating The chemical and electrical activities that make life possible are controlled by a constellation of ion channels. These are transmembrane proteins that regulate the differential concentration of ions across the cell membrane, with, in the case of K+ channels, the extraordinary ability to differentiate between extremely similar ions such as Na+ and K+. Although ion selectivity and gating mechanisms have been studied for many years, the underlying structural temporal dynamics remain poorly understood. Using techniques such as single molecule FRET (smFRET) or fluorescent non-canonical amino acids incorporation, it is possible to assess ion channel intramolecular movements and states in real time. In this project, I intend to incorporate a wide range of techniques, from electrophysiology to single molecule approaches to delineate the real-time dynamics of K+ channels in synthetic and live cell membranes. By combining my prior experience in ion channel biophysics and electrophysiology, along with establishing new approaches and training in relevant techniques, including smFRET, hyperspectral imaging, structure determination and Molecular Dynamic (MD) simulations, I propose two specific aims to address the unifying question: What are the structural dynamics of ion selectivity and gating in K+ channels? These studies will, first, test the hypothesis that the potassium channel selectivity filter (SF) with sequence TxGYG becomes dynamic and unstable in the absence of K+, acquiring more stable conformations in the presence of K+, using NaK/NaK2K (non-selective and K+ selective) channels as models. This aim, giving new insights about ion channel selectivity, will be completed during the K99 phase. Development of additional techniques and theoretical skills will allow me to broaden my efforts in the R00 phase to the study of structural dynamics of channel gating mechanisms in more complex channels, including the pharmacologically relevant eukaryotic Two-Pore-Domain K+ channel family (K2P) that contributes to leak currents in excitable and non-excitable tissues. This K+ channel family plays a critical role in a multitude of physiological and pathological conditions and understanding the gating and selectivity mechanisms will help to modulate their behavior to overcome different pathologies. Overall, the proposed integrated research and training will facilitate my becoming an independent researcher using a wide range of cutting-edge techniques to assist in the better understanding of membrane protein structural dynamics in health and disease.