Chemical tools for profiling and visualizing functioning ion channel complexes

描述（由申请人提供）： 描述（由申请人提供）： 摘要：此应用程序解决了广泛的挑战领域（06）使能技术和特定的挑战主题，06-GM-102：化学家/生物学家合作促进工具开发。该项目的目标是开发一套新的分子试剂，将化学探针传递到在活细胞中起作用的特定离子通道复合物。这项新技术的发展将使亚基组成和膜定位的离子通道复合物在天然细胞中的容易确定。对于这个项目，我们将靶向心肌细胞中发现的两个K+通道的离子传导和调节亚基上的N-连接聚糖：Kv 11 K+通道和KCNE I型跨膜肽。这个项目有三个目标。在目的1中，我们将衍生两种肽毒素，使得当它们结合到它们的同源K+通道复合物时，它们将用生物素或荧光探针共价标记复合物。衍生肽毒素的标记效率和特异性将在电记录、生化测定和荧光成像实验中得到证明。目的2和3将通过鉴定与心脏K+通道共组装的天然调节性KCNE亚基并确定活心肌细胞中K+通道的细胞表面定位和周转来证明衍生毒素组的效用。离子通道作为由膜包埋的离子传导和调节亚基组成的大分子蛋白质复合物发挥功能。复杂的组装对于适当的细胞功能至关重要，因为阻止共组装的突变会引起神经系统、心脏、肌肉和呼吸系统疾病。该项目描述了一套新试剂的开发，以探测健康和患病离子通道复合物的结构，功能和细胞定位。

DESCRIPTION (provided by applicant): DESCRIPTION (provided by applicant): Abstract: This application addresses broad Challenge Area (06) Enabling Technologies and specific Challenge Topic, 06-GM-102: Chemist/Biologist collaborations facilitating tool development. The goal of this project is to develop a new set of molecular reagents that deliver chemical probes to specific ion channel complexes functioning in living cells. The development of this novel technology will enable the facile determination of the subunit composition and membrane localization of ion channel complexes in native cells. For this project, we will target the N-linked glycans on ion- conducting and regulatory subunits of two K+ channels found in cardiomyocytes: Kv11 K+ channels and KCNE type I transmembrane peptides. There are three aims of this project. In Aim 1, we will derivatize two peptide toxins such that when they bind to their cognate K+ channel complex they will covalently label the complex with either biotin or a fluorescent probe. The labeling efficiency and specificity of the derivatized peptide toxins will be demonstrated in electrical recordings, biochemical assays and fluorescence imaging experiments. Aims 2 and 3 will demonstrate the utility of the panel of derivatized toxins by identifying the native regulatory KCNE subunits co-assembled with cardiac K+ channels and determining the cell surface localization and turnover of K+ channels in living cardiomyocytes. Ion channels function as macromolecular protein complexes composed of membrane-embedded ion-conducting and regulatory subunits. Complex assembly is vital for proper cellular function, as mutations that prevent co-assembly give rise to neurological, cardiac, muscular and respiratory diseases. This project describes the development of a novel set of reagents to probe the structure, function and cellular localization of healthy and diseased ion channel complexes.