NOVEL ACCESSORY PROTEIN MODULATING CARDIAC K+ CHANNELS

新型辅助蛋白调节心脏 K 通道

• 批准号: 2679797
• 负责人: Barbara A Wible
• 金额: $ 23.52万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2679797
• 关键词: Xenopus oocyte; electrophysiology; heart cell; immunocytochemistry; intermolecular interaction; laboratory rat; molecular chaperones; molecular cloning; muscle cells; myocardium; northern blottings; nucleic acid sequence; potassium channel; protein binding; protein localization; protein structure function; radiotracer; tissue /cell culture; transfection; voltage gated channel; western blottings; yeast two hybrid system

Voltage-gated K+ channels (Kv) are important in the physiology of excitable and nonexcitable cells. In excitable cells, they contribute to the repolarization phase of the action potential, while in nonexcitable cells, they contribute to diverse processes such as volume regulation, hormone secretion, and activation by mitogens. K+ channels are major targets for drug treatment in a number of diseases including cardiac arrhythmias, hypertension, epilepsy, and cerebrovascular ischemia. With multiple Kv channel genes whose products may assemble as multisubunit heteromeric complexes, there may be hundreds of functionally distinct K+ channels. Given their great diversity and fundamental importance, the cellular mechanisms regulating their synthesis, assembly, and metabolism are of prime interest, but at present, almost entirely unknown. To begin to dissect these processes, we have used the yeast two-hybrid system to identify novel cytoplasmic molecules that interact with Kv channel proteins. We have cloned a novel gene encoding a Kv channel binding protein (KChAP, for K Channel Associated Protein) which modulates the expression of a subset of Kv channels in heterologous expression system assays. We hypothesize that KChAP acts as a molecular chaperone by binding transiently to Kv alpha- subunits during synthesis to promote efficient channel assembly. This proposal will focus on the characterization of the molecular mechanisms by which KChAP interacts with K+ channel subunits to enhance current expression at the cell surface. In specific aim 1, we will identify and localize KChAP-Kv channel complexes in heterologous expression systems to determine whether KChAP is attached to mature K+ channels at the cell surface, like Kv beta subunits, or whether it acts more like a true chaperone by binding transiently during early stages of channel assembly. Specific aim 2 will identify the protein domains on KChAP and Kv channels that mediate functional interactions using a combination of biochemical, electrophysiological, and immunocytochemical methods. With these techniques, we will investigate the molecular mechanisms by which KChAP increases Kv currents. The cellular location of KChAP and its relationship to Kv alpha-subunits in native tissue, with an emphasis on heart, will be pursued in specific aim 3. Finally, specific aim 4 will identify other cellular protein partners of KChAP using multiple protein interaction cloning strategies. Together these studies should provide valuable information on a novel mechanism of K+ channel regulation.

电压门控 K+ 通道 (Kv) 在生理学中很重要 兴奋性细胞和非兴奋性细胞。 在兴奋细胞中，它们有助于 到动作电位的复极阶段，而在 非兴奋性细胞，它们有助于不同的过程，例如体积 调节、激素分泌和有丝分裂原的激活。 K+频道 是许多疾病药物治疗的主要靶点，包括 心律失常、高血压、癫痫、脑血管病 缺血。 具有多个Kv通道基因，其产物可组装 作为多亚基异聚复合物，可能有数百个 功能不同的 K+ 通道。 鉴于其巨大的多样性和 至关重要的是，调节其的细胞机制 合成、组装和代谢是人们最感兴趣的，但 存在，几乎完全未知。 为了开始剖析这些过程， 我们使用酵母双杂交系统来鉴定新的细胞质 与 Kv 通道蛋白相互作用的分子。 我们克隆了一个 编码 Kv 通道结合蛋白（KChAP，K 通道 相关蛋白）调节 Kv 子集的表达 异源表达系统检测中的通道。 我们假设 KChAP 通过瞬时结合 Kv α- 充当分子伴侣 合成过程中的亚基以促进有效的通道组装。 这 提案将重点关注分子机制的表征 KChAP 通过与 K+ 通道亚基相互作用来增强电流 表达于细胞表面。 在具体目标 1 中，我们将确定并 在异源表达系统中定位 KChAP-Kv 通道复合物 确定 KChAP 是否附着在细胞的成熟 K+ 通道上 表面，如 Kv beta 亚基，或者它的行为是否更像真实的 在通道早期阶段通过短暂结合形成伴侣 集会。 具体目标 2 将识别 KChAP 上的蛋白质结构域和 Kv 通道使用以下组合来介导功能交互 生物化学、电生理学和免疫细胞化学方法。 和 这些技术，我们将研究其分子机制 KChAP 增加 Kv 电流。 KChAP 的细胞定位及其 与天然组织中 Kv α 亚基的关系，重点是 心，将在具体目标 3 中追求。最后，具体目标 4 将 使用多种方法鉴定 KChAP 的其他细胞蛋白伙伴 蛋白质相互作用克隆策略。 这些研究共同应 提供有关 K+ 通道新颖机制的宝贵信息 规定。