Structural study of a Kv channel in different conformations in membranes

描述（由申请人提供）：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，在许多细胞事件中起关键作用。了解这些通道在膜中的运作方式不仅对阐明其功能的生物物理学意义重大，而且对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很重要。尽管过去对电压依赖性门控的结构和功能进行了广泛的研究，但关于电压依赖性门控的分子机制仍存在许多问题。最近的发现表明，电压门控钾（Kv）通道与磷脂膜有很强的相互作用，其功能受脂质环境的调节。但所有可用的电压门控离子通道结构都存在于去污剂或去污剂/脂质混合胶束中。我们假设，强蛋白质-脂质相互作用影响的电压门控离子通道的结构和功能，阐明的电压依赖性门控的结构细节需要在脂质双层的不同构象的电压门控离子通道的结构。在这个建议中，我们将使用作为一个模型系统的KvAP，从Aeropyrum pernix的Kv通道，通过表征在不同的脂质通道功能，并获得其结构在不同的构象来检查这一假设。KvAP是一个很好的模型系统，因为该蛋白质比重组真核细胞通道稳定得多，并且可以容易地重建到不同脂质组成的膜系统中。我们的目标1将专注于在与我们的二维结晶中使用的条件类似的条件下表征KvAP电压传感器的UP构象，然后获得这种构象中通道的结构。我们的目标2将使用生物化学和电生理学测定来表征有利于KvAP电压传感器处于其DOWN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。从我们的双管齐下的研究结果将提供新的证据来解决电压依赖性门控的基本问题。由于电压门控离子通道超家族中保守的结构特征，我们的研究结果将对真核细胞电压门控离子通道具有普遍意义。

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.