MECHANISMS OF THE VOLTAGE-DEPENDENT SODIUM CHANNEL

一个跨学科的方法将继续被用来阐明 电压依赖性的结构和分子机制 来自Electrophorus electricus electroplax钠通道。 的 这些研究的主要焦点将是非蛋白质的作用， 在功能中的事后获得域， 生物合成和钠通道的表达。 复溶 研究表明，碳水化合物去除和 脂质环境的变化将使用脂质 双层重组系统。 补充实验将使用 两栖类卵母细胞表达系统，以确定功能 翻译后修饰的后果 非蛋白质结构域的合成。 在生物合成研究中， 两栖类卵母细胞和鳗鱼电细胞将用于研究 加工过程中翻译后事件的性质和顺序 钠离子通道 感兴趣的将是糖基化，脂肪 酰化和高级结构域的发展， 这些事件发生的亚细胞区室。 为 这些研究将开发新的方法， 钠通道亚细胞合成器沿着 前体和评估通道的功能成熟 在合成过程中。在相关实验中， 翻译后修饰的靶向和表达 钠通道将使用特异性抑制剂进行研究， 生物合成 这些研究将利用单细胞 来研究原子核极化的机制， 合胞体细胞对直接通道差异调节 合成. 最后，通道肽的形貌将是 通过使用联合免疫学， 超微结构和生物化学方法。 地形 信息将通过有限的 蛋白水解，用抗体和凝集素进行蛋白质印迹分析，以及 一种新的电子显微镜抗体相关技术 与超微结构域结合。 总的来说，这些研究可能是 有望解决细胞如何调节的更普遍的问题， 钠通道功能的时间机制， 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.