MECHANISMS OF THE VOLTAGE-DEPENDENT SODIUM CHANNEL

将继续使用跨学科的方法来阐明 电压依赖性的结构和分子机制 钠离子通道来自于电子海绵。这个 这些研究的主要焦点将是非蛋白质的作用， 函数中的翻译后获得的域， 钠离子通道的生物合成和表达。在重建中 研究表明，碳水化合物去除和碳水化合物去除的功能效应 脂类环境的变化将使用脂类进行评估 双层重建体系。互补性实验将使用 一种确定两栖类卵母细胞功能的表达系统 翻译后药理改变的后果 非蛋白结构域的合成。在生物合成研究中，两者 两栖类卵母细胞和鳗鱼细胞将被用于研究 翻译后事件在加工过程中的性质和顺序 钠离子通道。令人感兴趣的是糖基化，脂肪 酰化，以及高阶结构域的发展和 这些事件发生的亚细胞隔间。为 这些研究将开发新的方法来分离 伴随着钠离子通道的亚细胞合成机械 并评估通道的功能成熟度。 在合成过程中。在相关实验中，它的作用是 靶向和表达中的翻译后修饰 将使用特定的钠通道抑制剂来研究钠通道 生物合成。这些研究将利用单个电细胞。 为了研究原子核极化的机制， 联会细胞被差异调节到直接经络 综合。最后，通道肽的拓扑图为 使用联合免疫学方法进行调查， 超微结构和生化方法。地形 信息将通过有限的组合方式获得 蛋白分解，用抗体和凝集素进行Western印迹分析，以及 一种利用电子显微镜关联抗体的新技术 结合到超微结构域。总的来说，这些研究可能是 预计将解决更普遍的问题，即细胞如何调节 钠通道功能和时间机制的研究进展 生物合成。

An interdisciplinary approach will continue to be used to elucidate the structrue and molecular mechanisms of the voltage-dependent sodium channel from Electrophorus electricus electroplax. The primary focus of these studies will be the role of nonprotein, post-translationally acquired domains in the function, biosynthesis, and expression of sodium channels. In reconstitution studies, the functional effects of both carbohydrate removal and changes in lipid environment will be assessed using the lipid bilayer reconstitution system. Complementary experiments will use an amphibian oocyte expression system to determine the functional consequences of pharmacologically altering post-translational synthesis of nonprotein domains. In biosynthetic studies, both amphibian oocytes and eel electrocytes will be used to study the nature and sequence of post-translational events in the processing of sodium channels. Of interest will be the glycosylation, fatty acylation, and development of higher order structural domains and the subcellular compartments in which these events occur. For these studies new methods will be developed both to fractionate the subcellular synthetic machinery along with sodium channel precursors and to assess the functional maturation of the channel during the synthetic process. In related experiments, the role of post-translational modification in the targeting and expression of sodium channels will be studied using specific inhibitors of biosynthesis. These studies will make use of single electrocytes to study the mechanisms by which nuclei in this polarized, syncitial cell are differentially regulated to direct channel synthesis. Lastly, the topography of the channel peptide will be investigated through the use of combined immunological, ultrastructural, and biochemical methods. Topographical information will be obtained with a combination of limited proteolysis, Western blot analysis with antibodies and lectins, and a novel technique using electeronmicroscopy to relate antibody binding to ultrastructural domains. Overall, these studies may be expected to address the more general question of how cells regulate the temporal mechanisms of sodium channel function and biosynthesis.