CHEMICAL SYNAPSES--BIOPHYSICAL STUDIES

本项目研究的是结构和功能之间的关系 对突触起重要作用的两类膜蛋白 变速箱。它使用了功能分析和表达技术。一个 第一组目标涉及神经递质门控通道，特别是 尼古丁AChR家族。研究将继续将域名本地化在 推测的胞外区对细胞的门控很重要 频道。这些研究始于嵌合克隆，表达于 并将进行定点突变。测试将会 继续使用与亲水序列相对应的合成肽 在受体内部，在卵母细胞中表达的受体。如果这些 序列对应于功能域，它们可能会影响通道 门控。将采用嵌合cDNA克隆(nAChR/GABA)进行定位 对电荷选择性很重要的区域(S)。非天然氨基酸 将使用合成的氨基酰化琥珀抑制剂引入nAChRs TRNAs。对这些受体的功能测量可能会揭示其位置 它们在配基结合和门控中很重要。循序渐进的方法 将被带到高分辨率的结构研究。这种方法依赖于 在确定膜蛋白的重要可溶域的基础上。 第二组目标涉及神经递质转运体， 特别是对GABA和其他氨基酸。克隆和 新的神经递质转运体家族成员的特征将 继续。电生理测量将继续在机械上进行 克隆和表达转运蛋白的研究。研究将继续进行 哺乳动物表达的转运蛋白的调控 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来定位 异源表达系统。随后将进行定点突变。 研究的受体和转运蛋白分子对健康至关重要 神经功能和构成许多临床靶点 重要的药物。

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.