Tuning neuronal excitability by axonal targeting of Kv7 channels

通过 Kv7 通道的轴突靶向调节神经元兴奋性

• 批准号: 9240673
• 负责人: Hee Jung Chung
• 金额: $ 33.92万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-05-15 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9240673
• 关键词: ANK3 gene; Action Potentials; Antiepileptic Agents; Axon; Axonal Transport; Binding; Biochemical; Biological Assay; Brain Diseases; C-terminal; Calmodulin; Chronic; Data; Dendrites; Development; Dimensions; Down-Regulation; Dynamin; Electrophysiology (science); Endocytosis; Endoplasmic Reticulum; Epilepsy; Epileptogenesis; Familial benign neonatal epilepsy; Fostering; Goals; Hippocampus (Brain); Human; Image; Impairment; Ion Channel; Kinesin; Knockout Mice; Laboratories; Link; Mediating; Methods; Molecular; Morphology; Mutation; Myokymia; Neuronal Plasticity; Neurons; Pathway interactions; Phosphatidylinositol 4,5-Diphosphate; Phosphorylation; Phosphorylation Site; Physiological; Play; Potassium Channel; Proteins; Publishing; Regulation; Research; Role; Seizures; Site; Surface; System; Tail; Temporal Lobe Epilepsy; Testing; Voltage-Gated Potassium Channel; Work; base; combat; density; epileptic encephalopathies; fluorescence imaging; immunocytochemistry; in vivo; insight; interdisciplinary approach; myosin VI; neuronal cell body; neuronal excitability; novel therapeutic intervention; public health relevance; syntaxin 1A; trafficking; voltage

 DESCRIPTION (provided by applicant): Epilepsy is caused by excessive neuronal excitability characterized by seizures, which are abnormal and uncontrolled discharges of action potentials. This common brain disorder is often caused by mutations in ion channels that form the basis for neuronal intrinsic excitability. Voltage-gated Kv7/KCNQ K+ channels potently inhibit repetitive and burst firing of action potentials. Kv7 activators are used as anti-epileptic drugs whereas mutations in Kv7.2 and Kv7.3 subunits cause epilepsy in humans. My work has shown that some epilepsy mutations impair polarized enrichment of Kv7 channels at the axonal surface, the subcellular domain governing action potential initiation and conduction, suggesting that their correct axonal targeting is critical for their physiologic function. However, the underlying mechanisms remain poorly understood. The goal of this proposal is to understand how axonal targeting of Kv7 channels is achieved and regulated by epilepsy mutations and neuronal activity, and how basal and regulated targeting impacts intrinsic excitability. This proposal is significant because dissecting these "unexplored" mechanisms will increase our understanding of the role of axonal Kv7 channels in regulating excitability, and foster the development of new therapeutic strategies for epilepsy that could enhance axonal surface density of Kv7. Given that chronic blockade of neuronal activity in hippocampus leads to temporal lobe epilepsy, characterizing the regulation of Kv7 axonal targeting by chronic activity blockade of hippocampal neurons may provide mechanistic insights into plasticity of intrinsic excitability and epileptogenesis. My work has shown that Kv7 enrichment at the axonal surface involves a region in the Kv7.2 C- terminal tail upstream of ankyrin-G binding domain. This "axon-targeting" domain contains phosphorylation sites and interacts with calmodulin, syntaxin 1A, AKAP79/150 and PIP2. In our preliminary studies, we discover that Kv7 axonal targeting is regulated by calmodulin-mediated exit from the endoplasmic reticulum, dynamin-mediated endocytosis, and phosphorylation of Kv7.2. We further show that homeostatic increase in excitability induced by chronic activity blockade accompanies reduction in Kv7 current, Kv7.3, and AKAP150. Based on these data, our hypothesis is that Kv7.2 interacting molecules and phosphorylation mediate basal and activity-regulated Kv7 axonal targeting by linking Kv7 to the core traffic machinery for polarized targeting. To test this hypothesis, Aim 1 will determine the traffic pathways that mediate Kv7 axonal targeting. Aim 2 will identify Kv7.2 interacting proteins and phosphorylation that bind to the core traffic machinery for polarized targeting. Aim 3 will characterize the regulation of Kv7 axonal targeting by chronic activity blockade. To execute these aims, we will use live imaging, immunostaining, binding assays, and electrophysiology in dissociated and organotypic hippocampal cultures, which preserve the functional and morphological polarity of neurons. We will also utilize epilepsy mutations, knock-out mice, and in vivo alterations of neuronal activity.

 描述（由申请人提供）：癫痫是由神经元过度兴奋引起的，其特征为癫痫发作，这是动作电位的异常和不受控制的放电。这种常见的脑部疾病通常是由离子通道突变引起的，离子通道是神经元内在兴奋性的基础。电压门控Kv 7/KCNQ K+通道有效抑制动作电位的重复和爆发放电。Kv 7激活剂被用作抗癫痫药物，而Kv7.2和Kv7.3亚基的突变导致人类癫痫。我的工作表明，一些癫痫突变损害了轴突表面Kv 7通道的极化富集，这是控制动作电位起始和传导的亚细胞结构域，这表明它们正确的轴突靶向对其生理功能至关重要。然而，对潜在的机制仍然知之甚少。该提案的目标是了解Kv 7通道的轴突靶向如何通过癫痫突变和神经元活动实现和调节，以及基础和调节靶向如何影响内在兴奋性。这一建议是重要的，因为解剖这些“未探索”的机制将增加我们对轴突Kv 7通道在调节兴奋性中的作用的理解，并促进癫痫的新治疗策略的发展，可以提高Kv 7的轴突表面密度。鉴于海马神经元活动的慢性阻断导致颞叶癫痫，表征海马神经元慢性活动阻断对Kv 7轴突靶向的调节可能为内在兴奋性和癫痫发生的可塑性提供机制见解。我的工作已经表明，轴突表面的Kv 7富集涉及Kv7.2 C末端尾中的锚蛋白-G结合结构域上游的区域。这个“轴突靶向”结构域含有磷酸化位点，并与钙调蛋白、突触融合蛋白1A、AKAP 79/150和PIP 2相互作用。在我们的初步研究中，我们发现，Kv 7轴突靶向调节钙调蛋白介导的退出内质网，动力蛋白介导的内吞作用，磷酸化Kv7.2。我们进一步表明，慢性活动阻滞诱导的兴奋性稳态增加伴随着Kv 7电流、Kv7.3和AKAP 150的减少。基于这些数据，我们的假设是，Kv7.2相互作用分子和磷酸化介导的基础和活性调节Kv 7轴突靶向连接Kv 7的核心交通机械极化靶向。为了验证这一假设，Aim 1将确定介导Kv 7轴突靶向的交通途径。目的2将确定Kv7.2相互作用蛋白和磷酸化结合的核心交通机械极化靶向。目的3将表征慢性活性阻断对Kv 7轴突靶向的调节。为了实现这些目标，我们将在分离的和器官型海马培养物中使用活体成像、免疫染色、结合测定和电生理学，这些培养物保留了神经元的功能和形态极性。我们还将利用癫痫突变、基因敲除小鼠和体内神经元活动的改变。