High Resolution Optical Analysis of Nav1.6 Localization, Trafficking and Function

描述（由申请人提供）：电压门控Na+ (Nav)通道启动神经系统中的大多数动作电位，α亚基突变负责从缺乏疼痛表型到癫痫的病理。轴突初始段（AIS）和Ranvier节点的高Nav通道密度被认为调节这些神经元室内的动作电位阈值。调控这些亚细胞结构域的机制，以及这种定位的功能后果，与人类健康直接相关，因为癫痫等疾病是由Na+通道运输或定位缺陷引起的。此外，缺血损伤后轴突初始段Na+通道定位的丧失可能导致神经元功能障碍，调节这种再定位可能是未来治疗中风的一种方法。热性惊厥可能起源于AIS，因为温度升高会增强AIS局部Nav通道的活性。有趣的是，创伤性脑损伤后AIS结构重塑，在中枢神经系统创伤模型中，Nav通道阻滞剂具有神经保护作用。尽管Nav通道在AIS中具有生理和病理意义，但关于这些通道的运输、维持和位置依赖功能的信息很少。事实上，该领域的主要争论集中在诸如导航如何将流量传输到AIS以及这些通道的功能百分比等问题上。由于缺乏荧光标记的导航通道结构和活细胞成像方法，这些领域的研究一直受到阻碍。本研究方案利用新型Nav通道构建，结合高分辨率单分子成像方法，实时监测海马神经元体细胞和AIS内Nav1.6通道的转运、定位和功能。特异性目的1将测试与Nav1.6在轴突初始段定位机制相关的假设。特异性目标2将研究Nav1.6通道活性如何随着细胞表面位置的变化而变化。这两个目标的共同假设都涉及到Nav1.6如何对类似缺血的神经元损伤做出反应。本研究将加深我们对神经元导航通道的功能和调控的认识。预防缺血性损伤引起的Nav通道定位改变是未来药物开发的潜在目标。

DESCRIPTION (provided by applicant): Voltage-gated Na+ (Nav) channels initiate the majority of action potentials in the nervous system and alpha subunit mutations are responsible for pathologies ranging from an absence of pain phenotype to epilepsy. A high Nav channel density at the axon initial segment (AIS) and node of Ranvier is believed to regulate action potential threshold within these neuronal compartments. Mechanisms regulating the targeting to these sub- cellular domains, and the functional consequences of such localization, are directly relevant to human health since diseases such as epilepsy are caused by Na+ channel trafficking or localization defects. In addition, loss of appropriate Na+ channel localization to te axon initial segment after ischemic injury likely contributes to neuronal dysfunction and modulation of this relocalization could be a future treatment for stroke. Febrile seizures may originate in the AIS since increased temperature enhances the activity of AIS localized Nav channels. Interestingly, the AIS is structurally remodeled following traumatic brain injury and in models of central nervous system trauma Nav channel blockers are neuro-protective. Despite the physiological and pathological significance of the Nav channels in the AIS, little information exists concerning the trafficking, maintenance, and location-dependent function of these channels. In fact, major debates in the field center around questions such as how Nav channels traffic to the AIS and what percentage of these channels are functional. Research in these areas has been hampered by a lack of fluorescently tagged Nav channel constructs and live cell imaging approaches. This research proposal utilizes novel Nav channel constructs in conjunction with high resolution single molecule imaging approaches to monitor the real-time trafficking, localization, and function of Nav1.6 channels in the soma and AIS of hippocampal neurons. Specific Aim 1 will test hypotheses relating to the mechanisms of Nav1.6 localization at the axon initial segment. Specific Aim 2 will examine how Nav1.6 channel activity varies as a function of cell surface location. Hypotheses common to both aims deal with how Nav1.6 responds to ischemia-like neuronal insults. The proposed research will enhance our understanding of the function and regulation of neuronal Nav channels. Preventing the altered Nav channel localization induced by ischemic insult is a potential target for future drug development.