Assembly and Trafficking of Cardiac K+ Channels

描述(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的持续时间中起着重要作用。我们假设，针对特定膜微域的通道及其在大分子复合体中的组织允许细胞提高其对细胞外信号的反应效率。本申请中提出的工作旨在了解Kv1.5和Kv2.1钾通道运输和周转的分子机制。我们建议对调节延迟整流钾通道Kv1.5功能的大分子复合体(通道小体)进行表征。我们将结合生化和分子工具、膜片钳分析和在体小鼠模型来追求以下特定目标：(1)确定Kv1.5多肽转运到小窝的机制，特别是表征Kv1.5与小窝蛋白-3(Cav-3)和SAP97之间的相互作用。(2)研究翻译后修饰对Kv1.5/Cav-3/SAP97大分子复合体的运输、组装和稳定性的影响。定位SAP97和Cav-3之间的相互作用部位，并证明这种相互作用有助于Kv1.5靶向小窝。(3)研究Kv2.1的N末端半胱氨酸及其翻译后修饰在调节Kv2.1的组装、运输和门控中的作用。本申请中概述的工作将极大地增加我们对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.