K+ channel protein complexes in auditory biology

描述（由申请人提供）：本研究的长期目标是阐明确保钾通道蛋白-蛋白复合物正确组装并维持耳蜗导管中K+稳态的分子和细胞机制。KCNQ 1-KCNE 1 K+通道复合物是内淋巴K+分泌进入耳蜗导管的唯一机制。KCNQ 1或KCNE中的基因突变破坏了复合物的组装、运输或功能，引起Jervell和Lange-Nielsen综合征，这是一种隐性形式的先天性听力损失，伴有晕厥发作。本申请旨在确定KCNQ 1-KCNE 1在内耳中组装和运输的基本机制。本申请有三个目的：（1）我们将利用生物化学和电生理学实验的组合来鉴定KCNQ 1-KCNE 1复合物的蛋白质-蛋白质界面线的残基;（2）我们将通过使用酶促去糖基化，膜分离，细胞表面标记方法和免疫荧光;（3）我们将研究Jervell和Lange-Nielsen综合征（JLNS）突变，该突变通过N-连接糖基化破坏复合物的组装和运输。为此，我们将研究N-连接的糖基化在KCNE 1生物合成，复杂的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KCNQ 1-KCNE相关疾病。

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.