FUNCTIONAL DOMAINS OF K+ CHANNEL SUBUNIT PROTEINS

神经元和身体的其他细胞的生理特性是 受钾通道的种类和丰度的强烈影响 在细胞中表达。大量的K+通道基因正在被 克隆了多个亚基K+的单亚基编码 通道蛋白，但这些亚单位蛋白是如何靶向形成 细胞中发现的各种K+通道蛋白。一个特定的问题 这些实验将解决的是亚基的初级结构 引导K~+通道组装形成电学性质 神经元。初步结果描述了识别出一种 出现的Shaker亚家族K+通道蛋白的结构域(T1结构域) 成为自组织的四聚中心；四聚是 被认为是K+形成K+通道的关键步骤 通道亚单位蛋白。该T1域似乎没有交互作用 与来自另一个基因亚家族的K+通道克隆。具体目标 这笔赠款包括：1)对Shaker亚家族的进一步描述 T1N-末端四聚结构域，2)其他结构域的鉴定 参与K+通道亚基多聚化，3)表征 T1域中的亚家族依赖差异及其检验 T1亚家族的特异性占主导地位，4)检测其作用 功能性K+通道形成过程中的T1结构域相容 蛋白质。实现这些特定目标的实验依赖于 中介绍的技术或方法。 初步结果或目前正在实验室中使用。这位将军 方法是克隆不同K+通道亚基的亚片段 被认为编码T1结构域的蛋白质，并检测它们的 功能和亚家族特异性。一系列的诱变实验 将确定关键的特定区域和氨基酸残基 针对T1结构域的四聚化和亚家族特异性。最后，一套 的嵌合K+通道亚单位蛋白将被创建，并交换T1 结构域，以测试T1在确定亚单位蛋白中的重要性 同、异多聚体K+的相互作用和形成 频道。因为K+通道蛋白是一种日益重要的 治疗哮喘、高血压、糖尿病、多发性硬化症药物的目标 硬化症，癫痫，以及潜在的许多其他疾病，这些 实验将通过提供关键的 K+通道亚单位蛋白的事件和结构域信息 参与形成离子通道的物质。

The physiological properties of a neuron and other cells of the body are strongly affected by the variety and abundance of potassium channels expressed in the cell. A large number of K+ channel genes are being cloned that apparently code for single subunits of multisubunit K+ channel proteins, but how are these subunit proteins targeted to form the variety of K+ channel proteins found in a cell. A specific question these experiments will address is whether subunit primary structures guide K+ channel assembly in forming the electrical properties of neurons. The Preliminary Results describe experiments that identify a domain of a Shaker Subfamily K+ channel protein (T1 domain) that appears to be a self organizing tetramerization center; tetramerization is thought to be a critical step in the formation of K+ channels by K+ channel subunit proteins. This T1 domain does not appear to interact with a K+ channel clone from another gene subfamily. The Specific Aims of this grant are: 1) Further Characterization of the Shaker Subfamily T1 N-Terminal Tetramerization Domain, 2) Identification of Other domains Involved in K+ Channel Subunit Multimerization, 3) Characterization of the Subfamily Dependent Differences in the T1 Domain and Testing Whether T1 Subfamily Specificity is Dominant, and 4) Examination of the Role of T1 Domain Compatibility in the Formation of Functional K+ Channel Proteins. The experiments to accomplish these Specific Aims rely exclusively on techniques or methods that are presented in the Preliminary Results or are being used currently in the lab. The general approach is to clone subfragments of different K+ channel subunit proteins that are thought to encode T1 domains, and to test for their function and subfamily specificity. A series of mutagenesis experiments will identify specific regions and amino acid residues that are critical for T1 domain tetramerization and subfamily specificity. Finally, a set of chimeric K+ channel subunit proteins will be created, with swapped T1 domains, to test for the importance of T1 in determining subunit protein interaction and formation of homomultimeric and heteromultimeric K+ channels. Because K+ channel proteins are an increasingly important target for drugs to treat asthma, high blood pressure, diabetes, multiple sclerosis, epilepsy and potentially a host of other disorders, these experiments will benefit health research by providing critical information about the events and domains of K+ channel subunit proteins that are involved in forming the ion channel.