K+ channel protein complexes in auditory biology

描述(申请人提供)：这项研究的长期目标是阐明确保钾通道蛋白-蛋白复合体正确组装和维持耳蜗管中K+动态平衡的分子和细胞机制。KCNQ1-KCNE1K+通道复合体是内淋巴K+分泌进入耳蜗管的唯一机制。KCNQ1或KCNE的基因突变会破坏复合体的组装、运输或功能，从而导致Jervell和Lange-Nielsen综合征，这是一种伴有突触发作的隐性先天性听力损失。这项应用旨在确定KCNQ1-KCNE1在内耳组装和运输的基本机制。这项应用有三个目标：(1)我们将利用生化和电生理实验相结合的方法来确定KCNQ1-KCNE1复合体蛋白质-蛋白质界面上的残基；(2)我们将通过利用酶脱糖基化、膜分离、细胞表面标记方法和免疫荧光来检测蛋白质的细胞转运模式，从而确定确保KCNE1与KCNQ1组装的细胞机制；(3)我们将研究Jervell和Lange-Nielsen综合征(JLNS)的突变，它通过N-连锁糖基化破坏了复合体的组装和运输。为此，我们将研究N-连接糖基化在KCNE1生物发生、复杂组装和细胞运输中的作用。这些目标的结果将提供一个分子和细胞框架，这将有助于理解JLNS和其他与KCNQ1-KCNE相关的疾病。

DESCRIPTION (provided by applicant): The long-term goal of this research is to elucidate the molecular and cellular mechanisms that ensure potassium channel protein-protein complexes properly assembly and maintain K+ homeostasis in the cochlear duct. The KCNQ1-KCNE1 K+ channel complex is the exclusive mechanism for endolymphatic K+ secretion into the cochlear duct. Genetic mutations in either KCNQ1 or KCNE that disrupt the assembly, trafficking or function of the complex give rise to Jervell and Lange-Nielsen Syndrome, a recessive form of congenital hearing loss accompanied with syncopal episodes. This application is directed at determining the basic mechanisms of KCNQ1-KCNE1 assembly and trafficking in the inner ear. There are three aims to this application: (1) we will identify the residues that line the protein-protein interface of the KCNQ1-KCNE1 complex utilizing a combination of biochemical and electrophysiological experiments; (2) we will determine the cellular mechanisms that ensure KCNE1 assembles with KCNQ1 by examining the cellular trafficking patterns of the proteins using enzymatic deglycosylation, membrane fractionation, cell surface labeling methods and immunofluorescence; (3) we will investigate a Jervell and Lange-Nielsen Syndrome (JLNS) mutation that disrupts assembly and trafficking of the complex via N-linked glycosylation. For this aim, we will examine the role of N-linked glycosylation in KCNE1 biogenesis, complex assembly and cellular trafficking. The results from these aims will provide a molecular and cellular framework, which will aid in the understanding of JLNS and other KCNQ1-KCNE-linked diseases.