CHEMICAL SYNAPSES--BIOPHYSICAL STUDIES

本项目研究结构与功能之间的关系， 这两类膜蛋白对突触的形成和突触的形成都很重要， 传输 它采用功能测定和表达技术。 一 第一组目标涉及神经递质门控通道，特别是 尼古丁AChR家族 研究将继续本地化领域， 假定的细胞外区域对于门控 频道。 这些研究开始于表达于大肠杆菌中的嵌合克隆。 卵母细胞，并将进行定点诱变。 测试将 继续合成对应于亲水序列的肽 在受体内，在卵母细胞中表达的受体上。 如果这些 序列对应于功能结构域，它们可以影响通道 门控 嵌合cDNA克隆（nAChR/GABA）将用于定位 对于电荷选择性重要的区域。 非天然氨基酸 将使用合成氨酰琥珀抑制剂引入nAChRs tRNA。 对这些受体的功能测量可以揭示 在配体结合和门控中很重要 一种渐进的办法 将进行高分辨率的结构研究 该方法依赖 在表征膜蛋白的重要可溶性结构域时。 第二组目标涉及神经递质转运蛋白， 特别是GABA和其他氨基酸。 克隆及 新的神经递质转运蛋白家族成员的特征将 继续 电生理测量将继续在机械 转运蛋白的克隆和表达研究。 研究将继续 对哺乳动物细胞中表达的转运蛋白的调节 细胞 转运蛋白上的重要功能域将是 通过测量嵌合转运蛋白的功能， 异源表达系统。 随后进行定点诱变。 研究的受体和转运分子对健康至关重要。 神经元的功能，并构成许多临床上的目标 重要药物。

This project studies the relationship between structure and function for two classes of membrane protein that are important for synaptic transmission. It employs functional assays and expression techniques. A first set of goals concern neurotransmitter-gated channels, particularly the nicotinic AChR family. Studies will continue to localize domains in the putative extracellular region that are important for the gating of the channel. These studies have begin with chimeric clones expressed in oocytes and will proceed to site-directed mutagenesis. Tests will continue with synthetic peptides corresponding to hydrophilic sequences within the receptors, on receptors expressed in oocytes. If these sequences correspond to functional domains, they may affect channel gating. Chimeric cDNA clones (nAChR/GABA) will be employed to localize the region(s) important for charge selectivity. Non-natural amino acids will be introduced into nAChRs using synthetic aminoacyl amber suppressor tRNAs. Functional measurements on these receptors may reveal positions that are important in ligand binding and gating. An incremental approach will be taken to high-resolution structural studies. The approach relies upon characterizing important soluble domains of the membrane proteins. A second set of goals concerns neurotransmitter transporters, particularly for GABA and other amino acids. Cloning and characterization of new neurotransmitter transporter family members will continue. Electrophysiological measurements will continue in mechanistic studies on the cloned and expressed transporters. Studies will continue on modulation and regulation of the transporters expressed in mammalian cells. Important functional domains on the transporters will be localized by measuring the function of chimeric transporters in heterologous expression systems. Site-directed mutagenesis will follow. The receptor and transporter molecules studied are crucial for healthy neuronal function and constitute the targets for many clinically important drugs.