Regulatory Mechanisms of Cardiac Repolarization

描述（由申请人提供）：电压门控K+通道（Kv通道） 在设定静息电位和确定 心血管系统中的复极。最近的证据表明 通常称为脂筏的专门微区存在于 大多数质膜的平面。这些结构域富含胆固醇， 鞘脂和集中了一些信号转导分子。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，靶向两种细胞中的脂筏。 异源表达系统与大鼠心血管系统和脑的关系。在 此外，Kv2.1和Kv1.5位于不同的筏舱中，Kv1.5 专门针对洞穴。证明了心脏筏含有 除了电压门控离子通道，我们还定位了 连接蛋白-43对成年心脏和培养的新生心肌细胞中的脂筏的影响。 细胞胆固醇的消耗改变了Kv2.1相关蛋白的浮力。 筏并将Kv2.1失活的中点移动近40 mV， 影响峰值电流密度或沟道激活。此外该 初步数据表明通道靶向和细胞表面定位 涉及到与特定的筏舱的关联。因此，木筏协会 从功能和本地化的角度来看都很重要。 本申请集中在Kv通道和连接蛋白之间的联系， 心血管系统。具体目标将1）表征Kv通道 与心脏和血管组织中脂筏的关联，2）解决 Kv1.5靶向小窝的机制，重点放在 α亚基组成和跨膜结构域，3）检查 Kv通道与脂筏结构域结合的功能意义，4） 确定筏完整性和间隙连接功能之间的关系， 在培养的心肌细胞中的定位，和5）分离单个心脏 包含Kv1.5和连接蛋白的闰盘区域，以鉴定 对通道功能和定位重要的相关蛋白质。给定 Kv通道和连接蛋白在心脏和血管中作用 平滑肌，拟议的研究将显着推进我们的 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.