MECHANISMS OF THE VOLTAGE-DEPENDENT SODIUM CHANNEL

电压依赖性钠通道的机制

• 批准号: 2262902
• 负责人: Simon R LEVINSON
• 金额: $ 25.85万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-01 至 1997-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2262902
• 关键词: Xenopus; Xenopus oocyte; alternatives to animals in research; exo alpha sialidase; glycoprotein biosynthesis; glycoprotein structure; glycosylation; laboratory rabbit; membrane biogenesis; membrane structure; molecular cloning; posttranslational modifications; protein structure function; sialate; site directed mutagenesis; sodium channel; transfection; voltage gated channel

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.

电压依赖的钠通道介导这一传播作用 神经和肌肉的潜能。从分子上讲，钠通道来自一种 许多组织由单个大的多肽组成，这是 被碳水化合物和疏水结构域严重修饰，推测是 脂类。这份申请书建议继续调查 这些非蛋白结构域在分子机制中的作用 下面是压敏门控和离子电导。 从重建研究中有强有力的初步证据表明 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，那么唾液酸残基的生物合成附着可以 代表一种自适应机制，允许某些细胞确定 适合于膜特性的通道选通特性和 该单元类型的功能要求。这一假设将是 使用互补的跨学科方法解决问题，在这种方法中 通道碳水化合物的组成将在以下方面进行操作 方法：1)通过从细胞表达的通道中去除糖 表面使用神经氨酸酶；2)抑制糖基化 利用选择性代谢抑制剂进行生物合成；3)表达 突变细胞系中缺乏各种元素的通道 糖基化机制，以及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，渠道 将cDNA导入细胞系及上述因素的影响 关于通道合成、表达和功能的操作将是 使用生化、免疫学和生物物理记录进行研究 技巧。对糖基化作用的进一步洞察将是 通过比较类似研究的结果获得 通道在非洲爪哇卵母细胞和哺乳动物肌肉纤维中表达。 总体而言，本应用程序中的研究将解决如何 渠道功能可以在翻译后的层面上进行修改。是这样的 信息可用于理解钠离子通道的作用 神经肌肉系统的退行性和发育性疾病 在设计更有效的麻醉和治疗药物方面 心律失常。