Biogenesis of Voltage-Gated K+ Channels

描述(由申请人提供)：这项建议旨在阐明电压门控K+通道(Kv)形成的分步机制以及获得二级、三级和四级Kv通道蛋白质结构所依据的特定蛋白质-蛋白质相互作用。Kv1.3是T淋巴细胞生理中重要的Kv通道，将是本研究中研究的主要通道。蛋白质折叠的决定因素不仅是理解生物发生的关键，也是运输机制的关键，因为最终保留/输出和降解信号是由蛋白质如何折叠/包装调节的。任何生物产生或运输步骤中的缺陷都会导致细胞表面Kv通道表达的改变。事实上，这些缺陷会导致病理，并可能是致命的。折叠和运输缺陷之间的联系可以推广到许多非通道疾病，并强调了澄清Kv生物发生中折叠和寡聚的基本原理的必要性。这就是我们提案的目标。总体目标分为六个具体目标。前四个目标致力于识别Kv13.3在生物发生过程中T1识别结构域和孔区的关键折叠和寡聚相互作用。第五个目标致力于评估何时以及在哪些隔室中实现了次生Kv构象。第六个目标致力于在组装过程中识别蛋白质-蛋白质、蛋白质-脂肪和蛋白质-水界面。本申请中提出的策略，即聚乙二醇化、质量标记和交联化，将使人们能够探索电子沉默残基在生物发生中的作用。此外，还将制作和研究Kv1.3的易位中间体。这些实验将采用一系列技术，包括对非洲爪哇卵母细胞和内质网微粒体膜进行生化和电生理分析。

DESCRIPTION (provided by applicant): This proposal is designed to elucidate the stepwise mechanisms of formation of voltage-gated K+ channels (Kv) and the specific protein-protein interactions that underlie the acquisition of secondary, tertiary, and quaternary Kv channel protein structure. Kvl.3, a Kv channel important in the physiology of T-lymphocytes, will be the primary channel studied in the proposed research. Determinants of protein folding are key to understanding not only biogenesis, but also trafficking mechanisms because ultimately the retention/export and degradation signals are modulated by how the protein is folded/packed. Defects in any biogenic or trafficking steps will result in altered expression of Kv channels at the cell surface. Indeed, these defects lead to pathology and can be lethal. The link between folding and trafficking defects is generalizable to many non-channel disorders and underscores the need to elucidate basic principles of folding and oligomerization in Kv biogenesis. This is the goal of our proposal. The overall goal is divided into six specific aims. The first four aims are devoted to identifying key folding and oligomerization interactions in the T1 recognition domain and the pore region of Kvl.3 during biogenesis. The fifth aim is devoted to assessing when and in which compartment secondary Kv conformations are achieved. The sixth aim is devoted to identifying protein-protein, protein-lipid, and protein-aqueous interfaces during assembly. The strategies proposed in this application, namely pegylation, mass-tagging, and crosslinking, will enable electrically silent residues to be probed for their role in biogenesis. Moreover, translocation intermediates of Kv 1.3 will be made and studied. The experiments will employ a range of techniques, including biochemical and electrophysiological assays in Xenopus oocytes and microsomal membranes of the endoplasmic reticulum.