Mechanisms of Kv1.2 regulation by tyrosine kinase

描述（由申请人提供）：我们研究的长期目标是了解控制神经元兴奋性的细胞机制。这一建议的重点是酪氨酸激酶依赖的抑制Kv1.2，电压门控钾通道。Kv1.2在整个神经和心血管系统中广泛表达，其抑制被认为在从癫痫和中风相关的神经元亢进到高血压相关的血管张力增加等人类病理中起关键作用。尽管它很重要，但Kv1.2抑制的机制仍然几乎完全未知。肌动蛋白细胞骨架似乎是Kv1.2调控的核心，因为肌动蛋白调控蛋白RhoA和cortinn结合Kv1.2并参与酪氨酸激酶对其的抑制。Kv1.2也经历酪氨酸磷酸化和肌动蛋白细胞骨架依赖的内吞作用。这表明通道抑制的物理机制涉及酪氨酸磷酸化依赖的Kv1.2与肌动蛋白细胞骨架相互作用的改变，导致通道内吞作用和随后的通道功能丧失。本研究的目标是了解酪氨酸激酶、动态肌动蛋白和内吞机制在Kv1.2聚集以调节细胞兴奋性的分子机制。为此，将使用一系列生化、分子生物学、免疫荧光显微镜和电生理学方法来解决以下具体目标：具体目标1：通过测试相关蛋白作为Kv1.2、肌动蛋白细胞骨架、酪氨酸激酶和参与内吞作用的蛋白质之间的物理通道的假设，确定肌动蛋白结合蛋白在Kv1.2调节中的作用。特定目的2：通过验证通道内单个酪氨酸在泛素依赖性通道内吞作用中具有特定和不同作用的假设，确定Kv1.2内吞作用的机制。具体目的3：通过验证RhoA与Kv1.2结合通过激活已知参与肌动蛋白丝重组的效应蛋白引起通道抑制的假设，阐明小g蛋白RhoA在Kv1.2抑制中的作用。这些目标共同探讨了酪氨酸激酶信号传导、细胞骨架生理和蛋白质运输在Kv1.2调控中协同作用的新观点。因此，这里提出的实验将从根本上为离子通道调节机制和神经元兴奋性的控制过程提供新的视角。

DESCRIPTION (provided by applicant): The long range objective of our research is to understand the cellular mechanisms governing neuronal excitability. This proposal focuses on the tyrosine kinase dependent suppression of Kv1.2, a voltage gated potassium channel. Kv1.2 is widely expressed throughout the nervous and cardiovascular systems and its suppression is hypothesized to have a key role in human pathologies ranging from neuronal hyperexcitability associated with seizure and stroke to increased vascular tone associated with hypertension. Despite its importance, the mechanism for Kv1.2 suppression remains almost completely unknown. The actin cytoskeleton appears to be central for Kv1.2 regulation since the actin-regulating proteins RhoA and cortactin bind to Kv1.2 and participate in its suppression by tyrosine kinases. Kv1.2 also undergoes tyrosine phosphorylation and actin cytoskeleton dependent endocytosis. This suggests a model in which the physical mechanism of channel suppression involves tyrosine phosphorylation dependent alteration of Kv1.2 interaction with the actin cytoskeleton leading to channel endocytosis and consequent loss of channel function. The goal of this proposal is to understand the molecular mechanisms by which tyrosine kinases, dynamic actin and the endocytotic machinery converge at Kv1.2 to regulate cellular excitability. To do so, a range of biochemical, molecular biological, immunofluorescence microscopy and electrophysiology methods will be used to address the following specific aims: Specific Aim 1: Determine the role of the actin binding protein cortactin in the regulation of Kv1.2 by testing the hypothesis that cortactin acts as a physical conduit between Kv1.2, the actin cytoskeleton, tyrosine kinases and proteins involved in endocytosis. Specific Aim 2: Determine the mechanisms of Kv1.2 endocytosis by testing the hypothesis that individual tyrosines within the channel have specific and divergent roles in ubiquitin dependent channel endocytosis. Specific Aim 3: Elucidate the role of the small G-protein RhoA in Kv1.2 suppression by testing the hypotheses that RhoA bound to Kv1.2 evokes channel suppression by activating effector proteins known to participate in actin filament reorganization. Collectively these aims explore the novel idea that tyrosine kinase signaling, cytoskeletal physiology and protein trafficking act as coordinated players in the regulation of Kv1.2. Thus, the experiments proposed here will provide fundamentally new perspectives into the mechanisms of ion channel regulation and the processes governing neuronal excitability.