NOVEL ACCESSORY PROTEIN MODULATING CARDIAC K+ CHANNELS

新型辅助蛋白调节心脏 K 通道

• 批准号: 6030907
• 负责人: Barbara A Wible
• 金额: $ 24.23万
• 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/6030907
• 关键词: Xenopus oocyte; cardiac myocytes; electrophysiology; immunocytochemistry; intermolecular interaction; laboratory rat; molecular chaperones; molecular cloning; 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.

电压门控性K+通道（Kv）在心脏的生理学中是重要的。 可兴奋和不可兴奋的细胞。 在易兴奋细胞中， 动作电位的复极相，而在 非兴奋性细胞，它们有助于不同的过程，如体积 调节、激素分泌和有丝分裂原激活。 k+通道 是许多疾病药物治疗的主要靶点， 心律失常、高血压、癫痫和脑血管疾病 缺血 有多个KV通道基因，其产物可以组装 作为多亚基异聚复合物，可能存在数百种 功能不同的K+通道。 鉴于其多样性和 至关重要的是，细胞机制调节其 合成、组装和代谢是主要的兴趣，但是， 目前，几乎完全未知。 为了开始剖析这些过程， 我们已经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 一种新的编码Kv通道结合蛋白（KChAP，用于K通道）的基因 相关蛋白），其调节Kv 异源表达系统测定中的通道。 我们假设 KChAP作为分子伴侣通过瞬时结合Kv α- 在合成过程中的亚基，以促进有效的通道组装。 这 该提案将侧重于分子机制的表征 KChAP通过与K+通道亚基相互作用增强电流 在细胞表面表达。 在具体目标1中，我们将确定和 在异源表达系统中定位KChAP-Kv通道复合物 确定KChAP是否附着于细胞的成熟K+通道， 表面，像Kv β亚基，或者它是否更像一个真正的 通道早期瞬时结合伴侣蛋白 组装件. 具体目标2将鉴定KChAP上的蛋白质结构域， KV通道，其使用以下组合介导功能性相互作用： 生物化学、电生理学和免疫细胞化学方法。 与 这些技术，我们将研究的分子机制， KChAP增加Kv电流。 KChAP的细胞定位及其在细胞内的表达 与天然组织中Kv α亚基的关系，重点是 心，将在具体目标3追求。 最后，具体目标4将 使用多重PCR鉴定KChAP的其他细胞蛋白伴侣 蛋白质相互作用克隆策略。 这些研究应 为钾离子通道的新机制提供了有价值的信息 调控