Structural study of a Kv channel in different conformations in membranes

描述(申请人提供)：膜中不同构象的Kv通道的结构研究电压门控离子通道是重要的膜蛋白，在许多细胞事件中发挥关键作用。了解这些通道是如何在膜上工作的，不仅对于阐明它们的功能是重要的，而且对于开发与各种电压门控离子通道功能障碍相关的人类疾病的药物治疗也是重要的。尽管过去对电压依赖门控的结构和功能进行了广泛的研究，但对于电压依赖门控的分子机制仍然存在疑问。最近的发现表明，电压门控性钾通道与磷脂膜有很强的相互作用，其功能受其脂质环境的调节。但所有可用的电压门控离子通道结构都存在于洗涤剂或洗涤剂/脂类混合胶束中。我们假设强烈的蛋白质-脂质相互作用影响电压门控离子通道的结构和功能，并且阐明电压依赖门控的结构细节需要在脂质双层中不同构象的电压门控离子通道的结构。在这项建议中，我们将使用来自Aeropyrum Pernix的Kv通道KvAP作为模型系统，通过描述不同脂质中的通道功能并获得其在不同构象中的结构来检验这一假设。KvAP是一个很好的模型系统，因为该蛋白比重组真核细胞通道更稳定，并且可以很容易地重组成不同脂组成的膜系统。我们的目标1将集中于表征KvAP电压传感器在类似于我们的二维结晶中使用的条件下的向上构象，然后在这样的构象中获得通道的结构。我们的目标2将使用生化和电生理分析来表征有利于KvAP电压传感器下行构象的条件，并将这些条件应用于筛选通道的2D晶体，这将为结构确定铺平道路。我们双管齐下的研究结果将为解决电压依赖门控的基本问题提供新的证据。由于电压门控离子通道超家族中保守的结构特征，我们的结果将对真核生物的电压门控离子通道具有普遍的指导意义。

DESCRIPTION (provided by applicant): Structural study of a Kv channel in different conformations in membranes Voltage-gated ion channels are important membrane proteins that play crucial roles in many cellular events. Understanding how these channels operate in membranes is important not only for the biophysical elucidation of their function but also for the development of pharmacological treatment for human diseases related to the dysfunction of various voltage-gated ion channels. Despite extensive structural and functional studies in the past, there are still open questions on the molecular mechanism of the voltage-dependent gating. Recent discoveries demonstrated that voltage-gated potassium (Kv) channels have strong interactions with phospholipid membranes, and their functions are modulated by their lipid environments. But all available structures of voltage-gated ion channels are in detergents or mixed detergent/lipid micelles. We hypothesize that the strong protein-lipid interactions affect both the structure and function of voltage-gated ion channels, and that elucidating the structural details of the voltage-dependent gating requires structures of a voltage-gated ion channel in different conformations in lipid bilayers. In this proposal, we will use as a model system the KvAP, a Kv channel from Aeropyrum pernix, to examine this hypothesis by characterizing the channel functions in different lipids and obtaining its structures in different conformations. The KvAP is a good model system because the protein is much more stable than recombinant eukaryotic channels, and can be readily reconstituted into membrane systems of different lipid composition. Our Aim 1 will focus on characterizing the UP conformation of the KvAP voltage sensor in conditions similar to those used in our two- dimensional crystallization, and then obtaining the structure of the channel in such a conformation. Our Aim 2 will use biochemical and electrophysiological assays to characterize conditions that favor the KvAP voltage sensor in its DOWN conformation, and apply these conditions to screen for 2D crystals of the channel, which will pave the way towards structure determination. Results from our two-pronged studies will offer new evidence to address the fundamental questions on voltage-dependent gating. Because of the well-conserved structural features in the superfamily of voltage-gated ion channels, our results will have general implications for eukaryotic voltage-gated ion channels.