Assembly and Trafficking of Cardiac K+ Channels

描述（由申请人提供）：在可兴奋细胞中，电压门控钾通道在调节动作电位持续时间方面发挥重要作用。我们假设，特定的膜微区和它们的组织在大分子复合物的通道的目标，使细胞增加其响应细胞外信号的效率。本申请中提出的工作是针对理解Kv1.5和Kv2.1钾通道的贩运和周转的分子机制。我们建议表征大分子复合物（通道体），其调节延迟整流钾通道Kv1.5的功能。我们将结合生物化学和分子生物学工具、膜片钳分析和体内小鼠模型来实现以下具体目标：（1）确定Kv1.5多肽向小窝运输的调控机制，特别是表征Kv1.5与小窝蛋白-3（Cav-3）和SAP 97之间的相互作用。(2)目的研究翻译后修饰对Kv1.5/Cav-3/SAP 97大分子复合物的运输、组装和稳定性的影响。定位SAP 97和Cav-3之间的相互作用位点，并证明这种相互作用有助于Kv 1.5靶向小窝。(3)描述Kv2.1的N-末端半胱氨酸及其翻译后修饰在调节Kv2.1组装、运输和门控中的作用。本申请中概述的工作应大大增加我们对Kv1.5和Kv2.1表达调控的理解。

DESCRIPTION (provided by applicant): In excitable cells, voltage-gated potassium channels play an important role in regulating the duration of the action potential. We hypothesize that the targeting of channels to particular membrane microdomains and their organization in macromolecular complexes allow cells to increase the efficiency of their response to extracellular signals. The work proposed in this application is directed towards understanding the molecular mechanisms underlying the trafficking and turnover of Kv1.5 and KV2.1 potassium channels. We propose to characterize the macromolecular complex (channelosome) that modulates the function of a delayed rectifier potassium channel, Kv1.5. We will use a combination of biochemical and molecular tools, patch-clamp analyses, and in vivo mouse models to pursue the following specific aims: (1) To determine the mechanisms that regulate trafficking of Kv1.5 polypeptide to caveolae, specifically to characterize the interactions between Kv1.5 and caveolin-3 (Cav-3) and SAP97. (2) To examine the role of posttranslational modifications on the trafficking, assembly, and stability on Kv1.5/Cav-3/SAP97 macromolecular complex. To map the site of interactions between SAP97 and Cav-3 and prove that this interaction contributes to the targeting of Kv1.5 to caveolae. (3) To characterize the role of N-terminal cysteines of Kv2.1 and their posttranslational modifications in regulating the assembly, trafficking, and gating of Kv2.1. The work outlined in this application should greatly increase our understanding of the regulation of expression of Kv1.5 and Kv2.1.