Mechanisms of Kv1.2 regulation by tyrosine kinase

酪氨酸激酶调节Kv1.2的机制

• 批准号: 6949663
• 负责人: ANTHONY D MORIELLI
• 金额: $ 35.03万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-15 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6949663
• 关键词: G protein; Xenopus; actin binding protein; actins; biological signal transduction; cytoskeleton; electrophysiology; endocytosis; enzyme mechanism; immunofluorescence technique; molecular biology; phosphorylation; potassium channel; protein protein interaction; protein structure function; protein transport; protein tyrosine kinase; tissue /cell culture; tyrosine; ubiquitin; voltage gated channel

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.

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。该提案的重点是酪氨酸激酶依赖性抑制 Kv1.2（一种电压门控钾通道）。 Kv1.2 在整个神经和心血管系统中广泛表达，并且假设其抑制在人类病理学中具有关键作用，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 Kv1.2 抑制很重要，但其机制仍然几乎完全未知。肌动蛋白细胞骨架似乎是 Kv1.2 调节的核心，因为肌动蛋白调节蛋白 RhoA 和 cortactin 与 Kv1.2 结合并参与酪氨酸激酶对其的抑制。 Kv1.2 还经历酪氨酸磷酸化和肌动蛋白细胞骨架依赖性内吞作用。这表明通道抑制的物理机制涉及 Kv1.2 与肌动蛋白细胞骨架相互作用的酪氨酸磷酸化依赖性改变，导致通道内吞作用和随后的通道功能丧失。该提案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 处汇聚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实现以下具体目标： 具体目标 1：通过测试 cortactin 作为 Kv1.2、肌动蛋白细胞骨架、酪氨酸之间的物理管道的假设，确定肌动蛋白结合蛋白 cortactin 在 Kv1.2 调节中的作用 参与内吞作用的激酶和蛋白质。具体目标 2：通过测试通道内的各个酪氨酸在泛素依赖性通道内吞作用中具有特定且不同的作用的假设，确定 Kv1.2 内吞作用的机制。具体目标 3：通过测试与 Kv1.2 结合的 RhoA 通过激活已知参与肌动蛋白丝重组的效应蛋白来引发通道抑制的假设，阐明小 G 蛋白 RhoA 在 Kv1.2 抑制中的作用。这些目标共同探索了酪氨酸激酶信号传导、细胞骨架生理学和蛋白质运输在 Kv1.2 调节中协调发挥作用的新想法。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全新的视角。