STRUCTURAL, KINETIC, AND CELLULAR MAPPING OF THE NAT+ CH

NAT CH 的结构、动力学和细胞图谱

• 批准号: 3409347
• 负责人: KIMON J ANGELIDES
• 金额: $ 9.25万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 1990-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3409347
• 关键词: antibody; chemical group; conformation; cytoskeleton; electron microscopy; electrophysiology; fluorescent dye /probe; local anesthetics; membrane permeability; membrane structure; myelination; neural transmission; neuromuscular disorder; neurons; neuropharmacology; neurotoxins; radiotracer; spectrometry; stop flow technique; tissue /cell culture

In order to understand the physiological basis of impulse propagation in nerve and muscle, it is necessary to define the molecular and cellular characteristics of the sodium channel. The overall objectives of this research program are to elucidate the molecular organization of the Na+ channel, its mechanism of action and regulation, and its distribution and dynamics on the nerve cell surface. I. Molecular Structure of the Na+ Channel. We propose to chemically modify and electrophysiologically assess important functional groups of the purified and reconstituted Na+ channel protein to gain insight into those groups critical for ion selection and gating. Conformational states accessible to the channel for ion selection and gating will be done by placing spectroscopic probes at functional sites and measuring the relaxation properties of the channel during action potential propagation by simultaneous optical and electrical recordings. Delayed fluorescence and photobleaching will be applied to examine the rotational and lateral diffusion of the reconsituted and native Na+ channel with the goals of reconstructing in three-dimensions the molecular structure given our other data, and to define the interactions between channels and with other cellular components. II. Cellular Distribution and Mobility of Na+ Channels. Because the sodium channel system lends such unique excitability characteristics to nerve and muscle cell membranes, and since the regulation of the distribution and density is associated with pathological changes that accompany certain neuromuscular disorders, we wil continue to localize and quantitate sodium channels at specific sites on myelinated and demyelinated nerve fibers by immuno-fluorescence and -electron microscopy. To examine the mobility of Na+ channels during neuronal differentiation, fluorescence recovery after photobleaching will be used in an in vitro myelination system of purified spinal cord neurons and Schwann cells to elucidate mechanism in the plasma membrane organization of Na+ channels, and their interactions with cytoskeletal and/or extracellular elements important in maintaining the segregation of Na+ channels on the axon surface.

为了理解在脑内脉冲传播的生理基础， 神经和肌肉，有必要定义分子和细胞 钠离子通道的特征 这一总体目标 研究计划是阐明Na+的分子组织 渠道，其作用机制和调节，及其分布和 神经细胞表面的动力学。 I. Na+通道的分子结构。 我们建议用化学方法 和电生理学评估的重要功能组的 纯化和重组Na+通道蛋白，以深入了解这些 对离子选择和选通至关重要的基团。 构象状态 离子选择和门控通道的可访问性将通过 将光谱探针放置在功能位点并测量 在动作电位传播过程中通道的松弛特性， 同时进行光学和电子记录。 延迟荧光和 光漂白将被应用于检查旋转和横向 重建和天然Na+通道的扩散，目的是 在三维空间中重建分子结构， 数据，并定义通道之间的交互以及与其他 细胞成分。 二. Na+通道的细胞分布和移动性。 因为钠 通道系统将这种独特的兴奋性特征赋予神经， 肌肉细胞膜，由于分布和 密度与病理变化有关， 神经肌肉疾病，我们将继续定位和定量钠 在有髓鞘和脱髓鞘的神经纤维上的特定部位的通道， 免疫荧光和电子显微镜。 检查的流动性 神经元分化过程中的Na+通道， 光漂白将被用于纯化的体外髓鞘形成系统中。 脊髓神经元和雪旺细胞，以阐明血浆中的机制 Na+通道的膜组织及其与 细胞骨架和/或细胞外成分，这些成分在维持 轴突表面Na+通道的分离。