Structural study of a Kv channel in different conformations in membranes

膜中不同构象的 Kv 通道的结构研究

• 批准号: 7866847
• 负责人: Qiu-Xing Jiang
• 金额: $ 30.12万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7866847
• 关键词: Address; Aeropyrum; Affect; Affinity; Binding; Biochemical; Biological Assay; Biological Models; Complex; Crystallization; DNA Sequence Rearrangement; Data; Detergents; Development; Dimensions; Environment; Event; Functional disorder; Gated Ion Channel; Image; Immunoglobulin Domain; Ions; Lateral; Lead; Lipid Bilayers; Lipids; Maps; Membrane; Membrane Lipids; Membrane Proteins; Methods; Micelles; Molecular; Molecular Conformation; Motion; Peptide Phage Display Library; Peptides; Pharmacological Treatment; Phospholipids; Physiological; Play; Potassium; Proteins; Recombinants; Resolution; Roentgen Rays; Role; Shapes; Structure; System; Toxin; density; human disease; improved; inorganic phosphate; insight; novel; public health relevance; reconstitution; sensor; two-dimensional; voltage

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. PUBLIC HEALTH RELEVANCE: Structural study of a Kv channel in different conformations in membranes Voltage-gated ion channels play essential roles in many physiological activities. They have been shown to require phospholipid bilayers for their proper function. Their strong interactions with phospholipids suggest that structures of the channel proteins in membranes are necessary for elucidating the structural details on voltage-dependent gating. In this proposal, a voltage-gated potassium (Kv) channel will be studied in two explicitly different conformations in membranes. Structural results will provide novel insights on how the voltage-gated ion channels are structurally adapted to their native lipid environment and how they rearrange themselves to achieve voltage-dependent gating.

描述（由申请人提供）：在膜电压门控离子通道中不同构象中的KV通道的结构研究是重要的膜蛋白，在许多细胞事件中起着至关重要的作用。了解这些通道在膜中的运作方式不仅对于其功能的生物物理阐明至关重要，而且对于与各种电压门控离子通道功能障碍相关的人类疾病的药理治疗的开发也很重要。尽管过去进行了广泛的结构和功能研究，但仍然关于电压依赖性门的分子机理的开放问题。最近的发现表明，电压门控钾（KV）通道与磷脂膜具有很强的相互作用，其功能受脂质环境调节。但是电压门控离子通道的所有可用结构均为洗涤剂或混合洗涤剂/脂质胶束。我们假设强蛋白脂质相互作用会影响电压门控离子通道的结构和功能，并且阐明了依赖电压依赖性门控的结构细节，需要在脂质双层中不同构象中电压门控离子通道的结构。在此提案中，我们将用作模型系统KVAP（来自Aeropyrum Pernix的KV通道）来检查该假设，通过表征不同脂质中的通道函数并以不同构象获得其结构。 KVAP是一个很好的模型系统，因为该蛋白质比重组真核通道更稳定，并且可以轻松地重构为不同脂质组成的膜系统。我们的目标1将集中在与我们两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，然后以这种构型以这种构型获得通道的结构。我们的AIM 2将使用生化和电生理测定法来表征有利于KVAP电压传感器下构象中的条件，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。我们的两项研究的结果将提供新的证据，以解决有关电压依赖性门的基本问题。由于电压门控离子通道的超家族中保存良好的结构特征，我们的结果将对真核电压门控离子通道具有一般影响。 公共卫生相关性：在膜电压门控离子通道不同构象中的KV渠道的结构性研究在许多生理活动中起着至关重要的作用。已显示它们需要磷脂双层的正常功能。它们与磷脂的强烈相互作用表明，膜中通道蛋白的结构对于阐明依赖电压依赖性门的结构细节是必需的。在此提案中，将在两个明显不同的构象中研究一个电压门控钾（KV）通道。结构性结果将提供有关电压门控离子通道如何在结构上适应其天然脂质环境以及它们如何重新安排自身以实现电压依赖性门控的新见解。