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

Nav1.6 定位、流量和功能的高分辨率光学分析

• 批准号: 8613282
• 负责人: Michael M. TAMKUN
• 金额: $ 32.44万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-30 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8613282
• 关键词: Ablation; Action Potentials; Acute; Ankyrins; Area; Ataxia; Axon; Binding; Cell surface; Cells; Cessation of life; Cognitive; Defect; Development; Diffusion; Disease; Dyes; Epilepsy; Exposure to; Febrile Convulsions; Functional disorder; Future; Glucose; Glutamates; Health; Hippocampus (Brain); Human; Image; Injury; Ischemia; Lead; Life; Location; Maintenance; Measures; Mediating; Membrane; Modeling; Molecular; Monitor; Multiple Sclerosis; Mus; Mutation; Nervous System Physiology; Nervous System Trauma; Nervous system structure; Neuraxis; Neuronal Dysfunction; Neurons; Optics; Oxygen; Pain; Pathology; Phenotype; Physiological; Protein Isoforms; Ranvier' s Nodes; Regulation; Relative (related person); Research; Research Proposals; Resolution; Schizophrenia; Sodium; Stroke; Surface; Temperature; Testing; Time; Traumatic Brain Injury; Tremor; Vesicle; cellular imaging; channel blockers; density; deprivation; drug development; improved; mutant; neuronal cell body; novel; prevent; public health relevance; research study; response; single molecule; trafficking; voltage; voltage clamp

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.

描述（由申请方提供）：电压门控Na+（Nav）通道启动神经系统中的大多数动作电位，α亚单位突变导致从疼痛表型缺失到癫痫的病理。在轴突起始段（AIS）和朗维尔结处的高Nav通道密度被认为调节这些神经元隔室内的动作电位阈值。调节靶向这些亚细胞结构域的机制和这种定位的功能后果与人类健康直接相关，因为疾病如癫痫是由Na+通道运输或定位缺陷引起的。此外，缺血性损伤后轴突起始段的适当Na+通道定位的丧失可能导致神经元功能障碍，并且这种重新定位的调节可能是中风的未来治疗。热性惊厥可能起源于AIS，因为升高的温度增强了AIS局部Nav通道的活性。有趣的是，AIS在创伤性脑损伤后结构重构，并且在中枢神经系统创伤模型中，Nav通道阻断剂具有神经保护作用。尽管在AIS的Nav通道的生理和病理意义，很少有信息存在有关的交通，维护，和位置依赖的功能，这些渠道。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Nav通道构建体和活细胞成像方法，这些领域的研究受到阻碍。本研究计划利用新型Nav通道构建体结合高分辨率单分子成像方法来监测海马神经元索马和AIS中Nav1.6通道的实时运输、定位和功能。具体目标1将测试与Nav1.6定位在轴突初始段的机制相关的假设。具体目标2将研究Nav1.6通道活性如何随细胞表面位置而变化。两个目标共同的假设涉及Nav1.6如何响应缺血样神经元损伤。该研究将有助于我们更好地了解神经元Nav通道的功能和调控。预防由缺血性损伤诱导的改变的Nav通道定位是未来药物开发的潜在目标。