Targeting K+ Channels to Caveolae:Cardiovascular System

描述（由申请人提供）：拟议研究的长期目标是了解离子通道靶向心血管系统细胞中特定膜微区的机制以及这种精确亚细胞组织的功能意义。心脏和血管平滑肌的电和收缩特性都依赖于调节心脏动作电位和控制动脉张力的电压门控钾（Kv）通道的表达。大多数组织，甚至心血管系统内的单个细胞，表达属于一个或多个亚家族的多种Kv通道类型。重要的是，这些不同通道亚型的亚细胞定位对于适当的细胞功能和信号传导至关重要。在确定渠道目标、集群和锚定所涉要素方面取得了进展。然而，目前尚不清楚如何确定膜平面内通道复合物的数量和位置，或者这种区室化如何影响通道功能。历史上，K+通道的靶向和定位被认为主要涉及通道和含PDZ结构域的支架蛋白或肌动蛋白细胞骨架之间的蛋白质-蛋白质相互作用。然而，最近，我们第一次表明，Kv通道的目标专门的脂质微区内的质膜平面。我们建议，通道蛋白和这些“脂筏”微结构域之间的相互作用代表了一种新的机制，通过改变脂质含量的通道靶向和调节通道特性。我们已经开发了一个独特的模型系统，在该系统中，它是可能的，以证明Kv通道的异构体特异性本地化不同的脂筏人口，与新的发现，Kv1.5本地化的小窝。该系统提供了一个独特的机会，在体外研究通道靶向的基本机制。体内初步结果表明，Kv1.5被发现在筏馏分在大鼠心脏和主动脉。此外，Western印迹分析表明，Kv1.5的翻译后/共翻译修饰可能靶向通道蛋白的小窝微结构域。此外，初步的实验表明，筏脂质的耗尽改变Kv1.5通道功能，这些影响可能涉及通道/酪氨酸激酶相互作用的破坏。基于这些数据，我们建议研究以下具体目的：1）建立Kv1.5与血管平滑肌中脂筏微结构域的关联; 2）确定Kv1.5靶向脂筏和小窝的机制; 3）证明Kv1.5定位于小窝对于适当的通道功能和/或调节是必要的。这项研究将大大促进我们对心血管系统电兴奋性调节的理解。

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to understand the mechanisms by which ion channels are targeted to specific membrane microdomains in cells of the cardiovascular system and the functional significance of this precise subcellular organization. The electrical and contractile properties of both the heart and vascular smooth muscle are dependent on the expression of voltage-gated potassium (Kv) channels that regulate the cardiac action potential and control arterial tone. Most tissues, and even single cells within the cardiovascular system, express multiple Kv channel types belonging to one or more subfamilies. Importantly, the subcellular localization of these different channel isoforms is critical for proper cell function and signaling. Progress has been made in identifying elements involved in channel targeting, clustering and anchoring. However, it is not yet clear how the number and location of channel complexes within the plane of the membrane are determined or how this compartmentalization affects channel function. Historically, K+ channel targeting and localization was thought to involve primarily protein-protein interactions between channels and PDZ-domain-containing scaffolding proteins or the actin cytoskeleton. Recently however, we showed for the first time that Kv channels target to specialized lipid microdomains within the plane of the plasma membrane. We propose that interactions between channel protein and these "lipid raft" microdomains represent a novel mechanism of channel targeting and for modulating channel properties via alterations in lipid content. We have developed a unique model system in which it is possible to demonstrate isoform-specific localization of Kv channels to distinct lipid raft populations, with the novel finding that Kv1.5 localized to caveolae. This system offers a unique opportunity to study basic mechanisms of channel targeting in vitro. Preliminary results in vivo indicate that Kv1.5 is found in the raft fractions in both rat heart and aorta. In addition, Western blot analysis indicates that post/cotranslational modifications of Kv1.5 may target channel protein to caveolar microdomains. Furthermore, preliminary experiments indicate that depletion of raft lipid alters Kv1.5 channel function and that these effects may involve a disruption of channel/tyrosine kinase interaction. Based on these data, we propose to investigate the following Specific Aims: 1) Establish the association of Kv1.5 with lipid raft microdomains in vascular smooth muscle; 2) Determine the mechanisms by which Kv1.5 is targeted to lipid rafts and caveolae; 3) Demonstrate that the localization of Kv1.5 to caveolae is necessary for proper channel function and/or regulation. The proposed research will significantly advance our understanding of the regulation of electrical excitability in the cardiovascular system.