Biogenesis of Voltrage-Gated K+ Channels

描述（由申请人提供）：钾离子通道是四聚体膜蛋白，为钾离子在细胞膜疏水屏障上扩散提供高度选择性的通道。因此，它们的形成和生物物理特性对神经元兴奋性、激素分泌和肌肉收缩等过程至关重要。离子通道的形成包括单体通道亚基的生物发生，亚基组装成四聚体通道，以及通道运输到适当的细胞膜，在那里它发挥其功能作用。虽然成熟钾通道的结构和功能已经被广泛研究，但对通道生物发生的早期折叠事件知之甚少。翻译和折叠缺陷会对钾离子通道的组装、运输和功能产生下游影响，并成为病理基础。我们研究计划的长期目标是阐明电压门控钾（Kv）通道生物发生中翻译和蛋白质折叠的基本原理，包括核糖体-新生肽复合物中的折叠事件。该提案分为四个相互关联的项目，每个项目都有几个目标。项目1致力于了解T1结构域（对Kv通道的组装和靶向至关重要）如何在生物发生过程中穿过隧道并被说服折叠。这种发展的模式并不存在。我们研究的预期结果将填补这一空白。项目二绘制电压传感器（VS）形成的事件和位置。这些发现适用于所有电压门控通道和VS折叠缺陷的机制。项目III定义了孔隙形成的先决条件和孔隙结构缺陷，这些缺陷是导致通道疾病（如长QT2综合征）的运输受损的基础。项目IV将为核糖体-新生肽复合物的变构通信产生新的范式，挑战反驳肽隧道动力学重要作用的现有范式。我们的研究结果将提出一种新的技术来确定肽在隧道中延伸的运动速率。我们的研究结果不仅涉及Kv通道形成和Kv蛋白的细胞水平，而且还涉及更广泛的生物发生和蛋白质折叠问题，并将为合理设计Kv折叠缺陷的治疗方法提供范例。在这些拟议的研究中使用的许多方法和策略（例如，聚乙二醇化，连接到核糖体隧道的新生肽的分子内交联分析）都是在我的实验室引入和发展的，用于分析二级，三级和四级折叠事件。拟议的实验将使用一系列技术，包括Kv生物源中间体的新型生化微分析，非洲爪蟾卵母细胞的电生理学和计算分析。

DESCRIPTION (provided by applicant): Potassium channels are tetrameric membrane proteins that provide a highly selective conduit for potassium ions to diffuse across the hydrophobic barrier of cell membranes. As such, their formation and biophysical properties are critical for processes like neuronal excitability, secretion of hormones, and muscle contraction. The formation of ion channels includes biogenesis of monomeric channel subunits, assembly of subunits into the tetrameric channel, and trafficking of the channel to the appropriate cellular membrane where it performs its functional role. Although the structure and function of mature potassium channels have been studied extensively, little is known about the early folding events in channel biogenesis. Defects in translation and folding have downstream consequences for assembly, trafficking, and function of potassium channels, and underlie pathology. The long-term goal of our research program is to elucidate basic principles of translation and protein folding in the biogenesis of voltage-gated potassium (Kv) channels, including folding events in the ribosome-nascent peptide complex. This proposal is divided into four interrelated Projects, each having several Aims. Project I is devoted to understanding how the T1 domain, critical for assembly and targeting of Kv channels, moves through the tunnel and is persuaded to fold during biogenesis. No models of this progression exist. The expected outcomes of our studies will fill this gap. Project II maps the events and location of voltage- sensor (VS) formation. Thes findings bear on all voltage-gated channels and mechanisms for VS folding defects. Project III defines prerequisites for pore formation and the defects in pore architecture that underlie the impaired trafficking responsible for channel diseases, e.g., Long QT2 Syndrome. Project IV will generate new paradigms for allosteric communication in the ribosome-nascent peptide complex, challenge existing paradigms that refute the important role of peptide-tunnel dynamics. Our results will advance a new technology for determining rates of peptide movement in the tunnel during peptide elongation. The results of our studies bear not only on Kv channel formation and cellular levels of Kv protein, but also on broader issues in biogenesis and folding of proteins, and will provide a paradigm for rational design of therapeutics for Kv folding defects. Many of the methods and strategies used in these proposed studies (e.g., pegylation, intramolecular crosslinking assays of a nascent peptide attached to a ribosomal tunnel) were introduced and developed in my laboratory to assay secondary, tertiary, and quaternary folding events. The proposed experiments will use a range of techniques including novel biochemical micro-assays of Kv biogenic intermediates, electrophysiology of Xenopus oocytes, and computational analysis. PUBLIC HEALTH RELEVANCE: Formation of voltage-gated potassium channels includes synthesis of the channel protein, assembly of these proteins into multimeric channels, and delivery of these channels to the plasma membrane. Defects in any of these steps will result in altered expression of the channels at the cell surface, with pathological and often lethal consequences. Our studies of ion channel folding bear on basic principles of protein folding and pave the way for understanding the molecular basis of protein misfolding, a fundamental cause of many diseases.