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

为了了解脉冲传播的生理基础 神经和肌肉，有必要界定分子和细胞 钠离子通道的特性。这个项目的总体目标是 研究计划是为了阐明Na的分子组织 经络及其作用和调节机制，以及经络的分布和 神经细胞表面的动态变化。 一、钠通道的分子结构。我们建议对其进行化学修饰 和电生理学评估重要的官能团 纯化和重组钠通道蛋白以深入了解这些 对离子选择和选通至关重要的组。构象状态 可通过以下方式访问通道以进行离子选择和选通 将光谱探针放置在功能位置并测量 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 将应用光漂白来检查旋转和侧向 重组和天然钠通道的扩散，目标是 在三维中重建我们的另一半的分子结构 数据，并定义通道之间的交互以及与其他 蜂窝组件。 II.钠通道的细胞分布和迁移性。因为钠 经络系统将这种独特的兴奋性特征赋予神经和 肌肉细胞膜，以及自调控的分布和 密度与某些伴随某些疾病的病理改变有关 神经肌肉障碍，我们将继续定位和定量钠 有髓神经纤维和脱髓鞘神经纤维上特定部位的通道 免疫荧光和电子显微镜。考察……的机动性 NA通道在神经元分化过程中，荧光恢复后 将光漂白用于纯化的体外髓鞘形成系统 脊髓神经元和雪旺细胞在血浆中的作用机制 钠离子通道的膜结构及其与钠离子的相互作用 细胞骨架和/或细胞外元素对维持 轴突表面Na通道的分离。

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.