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部分动作电位，阿尔法亚单位突变导致从没有疼痛表型到癫痫的各种病理变化。轴突起始段(AIS)和Ranvier结点的高NAV通道密度被认为调节了这些神经元隔室内的动作电位阈值。调控这些亚细胞结构域靶向的机制以及这种定位的功能后果直接关系到人类健康，因为癫痫等疾病是由Na+通道运输或定位缺陷引起的。此外，脑缺血损伤后轴突起始段Na+通道定位的缺失可能是导致神经元功能障碍的原因之一，这种重新定位的调节可能是未来治疗中风的一种方法。热性惊厥可能起源于AIS，因为温度升高增强了AIS局部NAV通道的活性。有趣的是，在创伤性脑损伤后，AIS的结构发生了改变，在中枢神经系统损伤的模型中，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，该领域的主要争论围绕着导航频道如何向AIS发送流量以及这些频道中有多大比例起作用等问题。由于缺乏荧光标记的NAV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。这项研究计划利用新的NAV通道结构结合高分辨率单分子成像方法来实时监测海马神经元胞体和AIS中Nav1.6通道的运输、定位和功能。特定目标1将测试与Nav1.6在轴突初始段定位机制有关的假说。特定目标2将研究Nav1.6通道活动如何随细胞表面位置的变化而变化。这两个目标都有共同的假设，即Nav1.6如何应对像缺血一样的神经元侮辱。建议的研究将加深我们对神经元NAV通道的功能和调节的理解。预防缺血损伤引起的NAV通道定位改变是未来药物开发的潜在靶点。

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.