K+ channel complexes: assembly, trafficking and function

描述(申请人提供)：这项研究的长期目标是阐明确保钾通道与适当的膜嵌入配对蛋白组装以实现适当的生理功能的分子和细胞机制。KCNQ1 K+通道在从心脏动作电位的复极化到上皮细胞的盐和水运输等许多人类生理学领域发挥着关键作用。为了产生这些不同生理作用所需的不同K+电流，KCNQ1通道必须与KCNE家族的跨膜肽形成膜包埋的复合体。KCNQ1通道或KCNE多肽的基因突变扰乱了复合体的功能或降低了其细胞表面的表达，会导致心律失常和耳聋。这项建议旨在确定KCNQ1-KCNE组装和功能的基本机制。这项提议有两个目的：(1)我们将利用突变扰动分析和半胱氨酸特异性化学结合电生理测量来确定KCNQ1-KCNE组装和功能所需的分子决定因素；(2)我们将确定KCNQ1-KCNE1复合体的细胞稳定性和转运速率是否是其细胞表面优先表达的细胞机制。为此，我们将对蛋白质进行代谢和化学标记，并使用亚细胞膜分离来确定它们的细胞组装和区隔位置。这些目标的结果将提供一个分子和细胞框架，以帮助理解KCNE相关疾病，并为新的治疗方法提供潜在的靶点。此外，随着与KCNE多肽组装的K+通道列表的增加，这些结果将适用于从神经元到肌肉的所有电兴奋细胞。

DESCRIPTION (provided by applicant): The long-term goal of this research is to elucidate the molecular and cellular mechanisms that ensure potassium channels assemble with the appropriate membrane-embedded partnering proteins for proper physiological function. The KCNQ1 K+ channel is a key player in many areas of human physiology from repolarization of the cardiac action potential to salt and water transport in epithelia cells. In order to generate the diverse set of K+ currents needed for these varied physiological roles, KCNQ1 channels must form membrane-embedded complexes with the KCNE-family of transmembrane peptides. Genetic mutations in either the KCNQ1 channel or the KCNE peptide that perturb complex function or reduce its cell surface expression cause cardiac arrhythmias and deafness. This proposal is directed at determining the basic mechanisms of KCNQ1-KCNE assembly and function. There are two aims to this proposal: (1) we will identify the molecular determinants required for KCNQ1-KCNE assembly and function using mutagenic perturbation analysis and cysteine-specific chemistries in combination with electrophysiological measurements; (2) we will determine whether the cellular stability and trafficking rates of the KCNQ1-KCNE1 complex are the cellular mechanisms responsible for their preferential cell surface expression. For this aim, we will metabolically and chemically label the proteins and determine their cellular sites of assembly and compartmentalization using subcellular membrane fractionation. The results from these aims will provide a molecular and cellular framework to aid in the understanding of KCNE-related diseases as well as providing potential targets for new therapies. Furthermore, as the list of K+ channels that assemble with KCNE peptides grows, these results will be applicable to all electrically excitable cells from neurons to muscle.