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支配神经元兴奋性的细胞机制。本研究主要研究电压门控钾通道Kv1.2对酪氨酸激酶的依赖抑制作用。Kv1.2在神经系统和心血管系统中广泛表达，其抑制被认为在人类病理中起关键作用，从与癫痫和中风相关的神经元过度兴奋到与高血压相关的血管张力增加。尽管它很重要，但抑制Kv1.2的机制仍然几乎完全未知。肌动蛋白细胞骨架似乎是Kv1.2调控的中心，因为肌动蛋白调节蛋白RhoA和Cortactin与Kv1.2结合，并通过酪氨酸激酶参与抑制Kv1.2。Kv1.2还经历酪氨酸磷酸化和肌动蛋白依赖的细胞骨架内吞作用。这提示了一种模型，在该模型中，通道抑制的物理机制涉及依赖于酪氨酸磷酸化的Kv1.2与肌动蛋白细胞骨架的相互作用，导致通道内吞，从而导致通道功能的丧失。这项建议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处聚合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特定目标：特定目标1：通过验证Cortactin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调控中的作用。具体目标2：通过验证通道内的单个酪氨酸在泛素依赖的通道内吞中具有特定的和不同的作用的假设，确定Kv1.2内吞的机制。具体目标3：通过验证RhoA与Kv1.2结合通过激活已知的参与肌动蛋白细丝重组的效应蛋白而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。总而言之，这些目的是探索酪氨酸激酶信号、细胞骨架生理学和蛋白质运输在Kv1.2调控中扮演协调角色的新想法。因此，这里提出的实验将为离子通道调节机制和控制神经元兴奋性的过程提供根本新的视角。

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.