FUNCTION AND ASSEMBLY OF POTASSIUM CHANNELS

这项建议旨在研究Kv1.3的组装和功能的分子机制，Kv1.3是一种电压门控钾通道(Kv)，在T淋巴细胞的生理中很重要。Kv1.3是Kv通道的典型成员，Kv通道在兴奋性和非兴奋性细胞中都具有不同的关键作用。拟议的实验将采用广泛的技术，包括在异源表达系统(非洲爪哇卵母细胞、哺乳动物细胞和微粒体膜)中进行生化和电生理分析。第一个目的是阐明在Kv转变为功能同源异构体的组装过程中，亚基内部和亚基之间的相互作用表面。我们假设低聚中间体的构象变化是沿着组装途径产生的，其中一些是后续组装步骤的先决条件。我们将使用联想和半胱氨酸可及性分析以及显性负抑制来探索通道拓扑结构，并测试特定的候选相互作用位点。取代半胱氨酸的可及性将部分通过使用与聚乙二醇联用的半胱氨酸试剂进行质量标记来进行分析。第二个目标是研究被认为在组装和通道门控中发挥作用的带电氨基酸所起的功能作用。我们将使用半胱氨酸扫描方法在膜片钳记录中探索S2、S3和S4跨膜片段的电压依赖性构象变化。我们将检验S2和S3片段中带负电荷的残基影响S4片段的电压依赖构象的假设。

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.