NOVEL ACCESSORY PROTEIN MODULATING CARDIAC K+ CHANNELS

电压门控K+通道(Kv)在心脏生理学中起重要作用 可兴奋和不可兴奋的细胞。在可兴奋的细胞中，它们对 到动作电位的复极化阶段，而在 不可兴奋的细胞，它们参与了不同的过程，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目标，包括 心律失常、高血压、癫痫和脑血管疾病 缺血症。具有多个Kv通道基因，其产物可以组装 作为多亚单位的异构体，可能有数百种 功能上不同的K+通道。考虑到它们的巨大多样性和 最重要的是，调节它们的细胞机制 合成、组装和新陈代谢是最重要的，但在 现在，几乎完全不为人所知。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。我们已经克隆了一个 K通道编码Kv通道结合蛋白(KChAP)的新基因 相关蛋白)，它调节Kv的一个子集的表达 异源表达系统中的通道分析。我们假设 KChAP作为分子伴侣与Kvα瞬时结合- 合成过程中的亚基，以促进有效的通道组装。这 建议将重点放在分子机制的表征上 KChAP通过与K+通道亚基相互作用来增强电流 在细胞表面表达。在具体目标1中，我们将确定和 KChAP-Kv通道复合体在异源表达系统中的定位 确定KChAP是否附着在细胞的成熟K+通道上 表面，像Kv贝塔亚基，或者它的行为更像是真的 在通道的早期阶段瞬时结合的伴侣 集合。特定目标2将确定KChAP上的蛋白质结构域和 KV通道使用以下组合来调节功能交互 生化、电生理和免疫细胞化学方法。使用 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ITS 与天然组织中Kvα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 识别KChAP的其他细胞蛋白伙伴 蛋白质相互作用克隆策略。这些研究加在一起应该 为K+通道的一种新机制提供有价值的信息 监管。

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.