Biogenesis of Voltrage-Gated K+ Channels

描述（由申请人提供）：钾通道是一种四聚体膜蛋白，为钾离子扩散穿过细胞膜的疏水屏障提供了高度选择性的通道。因此，它们的形成和生物物理特性对于神经元兴奋性，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。翻译和折叠的缺陷对钾通道的组装、运输和功能具有下游后果，并且是病理学的基础。我们研究计划的长期目标是阐明电压门控钾（Kv）通道生物发生中的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I致力于了解T1结构域如何通过隧道移动，并在生物发生过程中被说服折叠，T1结构域对组装和靶向Kv通道至关重要。目前还没有这种进展的模型。我们研究的预期成果将填补这一空白。项目II绘制了电压传感器（VS）形成的事件和位置。这些发现与VS折叠缺陷的所有电压门控通道和机制有关。项目III定义了孔隙形成的先决条件和孔隙结构中的缺陷，这些缺陷是导致通道疾病的受损运输的基础，例如，长QT 2综合征。项目四将产生新的范例变构通讯的核糖体新生肽复合物，挑战现有的范例，驳斥了重要作用的肽隧道动力学。我们的研究结果将推进一种新的技术，用于确定在肽延伸过程中的隧道中的肽运动速率。我们的研究结果不仅涉及Kv通道的形成和Kv蛋白的细胞水平，而且涉及蛋白质的生物发生和折叠的更广泛问题，并将为合理设计Kv折叠缺陷的治疗方法提供范例。这些拟议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测定）被引入并在我的实验室中开发以测定二级、三级和四级折叠事件。拟议的实验将使用一系列的技术，包括新的生物化学微量测定KV生物中间体，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.