MECHANISMS OF THE VOLTAGE-DEPENDENT SODIUM CHANNEL

电压依赖性钠离子通道介导的传播行动 神经和肌肉的潜力。 分子上，钠离子通道来自 许多组织由单一的大多肽组成， 被碳水化合物和疏水结构域严重修饰， 脂质 本申请建议继续调查 这些非蛋白质结构域在分子机制中的作用， 是电压敏感门控和离子电导的基础。 重组研究有强有力的初步证据， 带负电荷的唾液酸残基附着在通道上 显著影响激活门控附近的局部电场 传感器. 如果是这样的话，那么唾液酸残基的生物合成附着可能 代表了一种适应性机制， 通道门控特性适合于膜特性， 这种细胞类型的功能需求。 这个假设将是 采用互补的跨学科方法， 通道碳水化合物组合物将在下面进行操作 途径：1）通过从细胞表达的通道中去除糖 2）使用神经氨酸酶抑制糖基化; 使用选择性代谢抑制剂进行生物合成; 3）表达 在突变细胞系中缺乏各种元件的通道， 糖基化机制，和4）克隆的通道cDNA的诱变， 修饰、移动或消除糖基化位点。 在这些研究中， 将cDNA转染到细胞系中，并观察上述影响。 对通道合成、表达和功能的操作将 使用生物化学、免疫学和生物物理学记录进行研究 技术. 进一步深入了解糖基化的作用将是 通过比较对以下动物进行的类似研究的结果获得： 在非洲爪蟾卵母细胞和哺乳动物肌纤维中表达。 总的来说，本申请中的研究将解决如何 通道功能可以在翻译后水平被修饰。 等 这些信息对于理解钠通道的作用有着重要的意义 神经肌肉系统的退化和发育障碍 以及设计更多用于麻醉的有效药物， 心律失常

Voltage-dependent sodium channels mediate the propagating action potential of nerve and muscle. Molecularly, sodium channels from a number of tissues are comprised of single large polypeptide that is heavily modified by carbohydrate and hydrophobic domains inferred to be lipid. This application proposes to continue investigations into the role of these nonprotein domains in the molecular mechanisms that underlie voltage-sensitive gating and ion conductance. There is strong preliminary evidence from reconstitution studies that negatively charged sialic acid residues attached to the channel significantly affect the local electrical field near an activation gating sensor. If so, then biosynthetic attachment of sialic acid residues may represent an adaptive mechanism that allows certain cells to determine channel gating characteristics appropriate to the membrane properties and functional requirements of that cell type. this hypothesis will be addressed using a complementary interdisciplinary approach in which channel carbohydrate compositions will be manipulated in the following ways: 1) through removal of sugars from channels expressed at the cell surface using neuraminidases; 2) inhibition of glycosylation during biosynthesis using selective metabolic inhibitors; 3) expression of channels in mutant cell lines lacking various elements of the glycosylation machinery, and 4) mutagenesis of cloned channel cDNAs to modify, move or eliminate glycosylation sites. In these studies channel cDNAs will be transfected into cell lines and the affect of the above manipulations on channel synthesis, expression, and function will be studied using biochemical, immunological, and biophysical recording techniques. Further insight into the roles of glycosylation will be obtained by comparing the results from similar studies performed on channels expressed in Xenopus oocytes and mammalian muscle fibers. Overall, the studies in this application will address the question of how channel function may be modified at the posttranslational level. Such information has applications to the understanding of sodium channel roles in degenerative and developmental disorders of the neuromuscular system and in the design of more affective drugs for use in anesthesia and cardiac arrhythmias.