FUNCTION AND ASSEMBLY OF POTASSIUM CHANNELS

钾通道的功能和组装

• 批准号: 2850552
• 负责人: Carol J Deutsch
• 金额: $ 50.74万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-05-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2850552
• 关键词: T lymphocyte; Xenopus oocyte; cell biology; conformation; cysteine; intermolecular interaction; membrane potentials; potassium channel; protein biosynthesis; protein structure function; tissue /cell culture; voltage /patch clamp

This proposal is designed to examine molecular mechanisms underlying the assembly and function of Kv1.3, a voltage-gated potassium channel (Kv) important in the physiology of T- lymphocytes. Kv1.3 is a typical member of Kv channels, which have diverse and critical roles in both excitable and non-excitable cells. The proposed experiments will employ a wide range of techniques, including biochemical and electrophysiological assays in heterologous expression systems (Xenopus oocytes, mammalian cells, and microsomal membranes). The first aim is to elucidate interaction surfaces, both within and between subunits, during the assembly of Kv changes into functional homotetramers. We hypothesize that conformational changes in oligomeric intermediates are generated along the assembly pathway, and that some are prerequisites for subsequent assembly steps. We will use association and cysteine accessibility assays and dominant negative suppression to explore channel topology and to test specific candidate sites of interaction. Substituted cysteine accessibility will be assayed in part by mass-tagging using a cysteine reagent conjugated with polyethylene glycol. The second aim is to study the functional role played by charged amino acids that are believed to play a role in both assembly and channel gating. We will explore voltage-dependent conformational changes of the S2, S3, and S4 transmembrane segments, using cysteine scanning methods in patch clamp recording. We will test the hypothesis that negatively charged residues in the S2 and S3 segments affect the voltage-dependent conformations of the S4 segment.

该提议旨在研究Kv1.3组装和功能的分子机制，Kv1.3是一种在T淋巴细胞生理学中重要的电压门控钾通道（Kv）。Kv1.3是Kv通道的典型成员，在兴奋性和非兴奋性细胞中具有不同的和关键的作用。 拟议的实验将采用广泛的技术，包括异源表达系统（非洲爪蟾卵母细胞，哺乳动物细胞和微粒体膜）的生化和电生理测定。第一个目的是阐明相互作用的表面，无论是内部和亚基之间，在组装过程中的KV变化成功能的同源四聚体。我们假设低聚物中间体的构象变化是沿着组装途径产生的，并且其中一些是后续组装步骤的先决条件。 我们将使用协会和半胱氨酸可及性测定和显性负抑制，探索通道拓扑结构和测试特定的候选网站的相互作用。 将使用与聚乙二醇偶联的半胱氨酸试剂通过质量标记部分测定取代的半胱氨酸可及性。第二个目的是研究被认为在组装和通道门控中起作用的带电氨基酸所起的功能作用。 我们将探讨电压依赖性的构象变化的S2，S3和S4跨膜段，在膜片钳记录中使用半胱氨酸扫描方法。 我们将检验S2和S3片段中带负电荷的残基影响S4片段的电压依赖性构象的假设。