Tuning neuronal excitability by axonal targeting of Kv7 channels

 描述(由申请人提供)：癫痫是由神经元过度兴奋引起的，以癫痫为特征，这是动作电位的异常和不受控制的放电。这种常见的大脑疾病通常是由离子通道突变引起的，离子通道是神经元固有兴奋性的基础。电压门控KV7/KCNQ K+通道有效地抑制动作电位的重复和突发性放电。Kv7激活剂被用作抗癫痫药物，而Kv7.2和Kv7.3亚单位的突变会导致人类癫痫。我的工作表明，一些癫痫突变损害了KV7通道在轴突表面的极化丰富，KV7通道是控制动作电位启动和传导的亚细胞区域，这表明它们正确的轴突靶向对它们的生理功能至关重要。然而，人们对其潜在机制仍知之甚少。这项建议的目的是了解KV7通道的轴突靶向是如何通过癫痫突变和神经元活动实现和调节的，以及基础靶向和调节靶向如何影响内在兴奋性。这一建议具有重要意义，因为解剖这些“未探索”的机制将增加我们对轴突KV7通道在调节兴奋性中的作用的理解，并促进癫痫新治疗策略的发展，从而提高KV7的轴突表面密度。鉴于海马神经元活动的慢性阻断会导致颞叶癫痫，表征海马神经元慢性活动阻断对KV7轴突靶向的调节可能为内在兴奋性的可塑性和癫痫的发生提供机械性的见解。我的工作表明，KV7在轴突表面的浓缩涉及到Kv7.2C末端尾部Ankyrin-G结合域上游的一个区域。这个“轴突靶向”结构域含有磷酸化位点，并与钙调蛋白、合成素1A、AKAP79/150和PIP2相互作用。在我们的初步研究中，我们发现KV7轴突靶向受到钙调蛋白介导的内质网退出、动力蛋白介导的内吞作用和Kv7.2的磷酸化的调节。我们进一步表明，慢性活动阻断引起的兴奋性的动态平衡增加伴随着KV7电流、Kv7.3和AKAP150的减少。基于这些数据，我们的假设是，Kv7.2相互作用的分子和磷酸化通过将KV7连接到核心交通机械来实现极化靶向，从而介导基础的和活性调节的KV7轴突靶向。为了验证这一假设，目标1将确定介导KV7轴突靶向的交通路径。AIM 2将鉴定Kv7.2相互作用蛋白和磷酸化，这些蛋白和磷酸化结合到核心交通机械上进行极化靶向。目的3研究慢性活动阻断对KV7轴突靶向的调节作用。为了实现这些目标，我们将在分离的和器官型的海马区培养中使用实时成像、免疫染色、结合分析和电生理学，以保持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变化。

 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.