Regulatory Mechanisms of Cardiac Repolarization

说明(申请人提供)：电压门控K+通道(Kv通道) 在静息电位的设定和决定中发挥重要作用 心血管系统的复极。最近的证据表明 专门的微域通常称为脂筏，存在于 大多数质膜的平面。这些区域富含胆固醇和 鞘磷脂和浓缩的一些信号转导分子。在 初步数据部分，我们证明了电压门控K+通道， Kv2.1、Kv1.1、Kv1.5和Kv1.4，而不是Kv4.2，都以脂筏为靶点 异源表达系统与大鼠心血管系统和脑。在……里面 此外，Kv2.1和Kv1.5驻留在不同的筏舱内，Kv1.5 特别针对洞穴。证明心脏筏含有 除了电压门控离子通道外，我们还定位了 连接蛋白-43与成人心脏和培养的新生心肌细胞中的脂筏。 细胞胆固醇的耗尽改变Kv2.1相关的浮力 将Kv2.1的失活中点漂移近40 mV 影响峰值电流密度或通道激活。此外， 初步数据显示通道定位和细胞表面定位 涉及与特定浮筏舱室的关联。因此，木筏协会 从功能和本地化的角度来看都很重要。 这个应用主要关注Kv通道和连接蛋白之间的联系 心血管系统。具体目标将1)描述Kv频道 与心脏和血管组织中的脂筏相关，2)解决 Kv1.5靶向小凹病毒的机制，重点是 关于α亚基组成和跨膜区，3)检查 Kv通道与脂筏结构域结合的功能意义 确定筏板完整性和缝隙连接功能之间的关系 在培养的心肌细胞中的定位；5)分离单个心脏 含有Kv1.5和连接蛋白的插入盘区以识别 与通道功能和定位有关的相关蛋白。vt.给出 Kv通道和连接蛋白在心脏和血管中的作用 这项拟议的研究将极大地推动我们的 了解心血管系统的电兴奋性。

DESCRIPTION (provided by applicant): Voltage-gated K+ channels (Kv channels) play an important role in setting the resting potential and determining repolarization in the cardiovascular system. Recent evidence suggests that specialized microdomains commonly referred to as lipid rafts exist within the plane of most plasma membranes. These domains are enriched in cholesterol and sphingolipids and concentrate a number of signal transduction molecules. In the Preliminary Data section, we demonstrate that the voltage-gated K+ channels, Kv2.1, Kv1.1, Kv1.5, and Kv1.4, but not Kv4.2, target to lipid rafts in both heterologous expression systems and the rat cardiovascular system and brain. In addition, Kv2.1 and Kv1.5 reside in different raft compartments, with Kv1.5 specially targeting to caveolae. Demonstrating that cardiac rafts contain proteins other than voltage-gated ion channels, we have also localized connexin-43 to lipid rafts in both adult heart and cultured neonatal myocytes. Depletion of cellular cholesterol alters the buoyancy of the Kv2.1-associated rafts and shifts the midpoint of Kv2.1 inactivation by nearly 40 mV without affecting peak current density or channel activation. In addition, the preliminary data suggest channel targeting and cell surface localization involve the association with specific raft compartments. Thus, raft association is significant from both a functional and localization standpoint. This application focuses on the link between both Kv channels and connexins in the cardiovascular system. The Specific Aims will 1) characterize Kv channel association with lipid rafts in cardiac and vascular tissue, 2) address the mechanisms involved in the targeting of Kv1.5 to caveolae, with emphasis placed on alpha subunit composition and transmembrane domains, 3) examine the functional significance of Kvchannel association with lipid raft domains, 4) determine the relationship between raft integrity and gap junction function and localization in cultured myocytes, and 5) isolate the individual cardiac intercalated disk regions containing Kv1.5 and connexins in order to identify the associated proteins important to channel function and localization. Given the role that both Kv channels and connexins play in cardiac and vascular smooth muscle, the proposed research will significantly advance our understanding of electrical excitability in the cardiovascular system.