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针对心血管系统细胞中特定的膜微域的机制以及这种精确的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依赖于电压门控钾(Kv)通道的表达，Kv通道调节心脏动作电位和控制动脉张力。大多数组织，甚至心血管系统中的单个细胞，都表达属于一个或多个亚家族的多种Kv通道类型。重要的是，这些不同通道异构体的亚细胞定位对于正常的细胞功能和信号转导至关重要。在确定涉及频道目标、集群和锚定的要素方面取得了进展。然而，目前还不清楚膜平面内通道复合体的数量和位置是如何确定的，或者这种划分是如何影响通道功能的。历史上，K+通道的靶向和定位主要涉及通道和含有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，我们首次发现Kv通道靶向于质膜平面内的特殊脂质微域。我们认为，通道蛋白和这些“脂筏”微域之间的相互作用代表了一种新的通道靶向机制，并通过改变脂质含量来调节通道特性。我们已经开发了一个独特的模型系统，在该系统中可以证明Kv通道对不同的脂筏群体具有异构体特异性的定位，新的发现是Kv1.5定位于小窝。该系统为研究体外通道靶向的基本机制提供了一个独特的机会。初步的体内实验结果表明，Kv1.5在大鼠心脏和主动脉的RAFT组分中均有表达。此外，Western印迹分析表明，Kv1.5的翻译后/共翻译修饰可能将通道蛋白靶向空泡微区。此外，初步实验表明，RAFT脂类物质的耗尽改变了Kv1.5通道的功能，这些影响可能涉及通道/酪氨酸激酶相互作用的中断。基于这些数据，我们建议研究以下特定目标：1)建立Kv1.5与血管平滑肌脂筏微域的联系；2)确定Kv1.5靶向脂筏和小窝的机制；3)证明Kv1.5定位于小窝是正确的通道功能和/或调节所必需的。这项拟议的研究将极大地促进我们对心血管系统中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.