MOLECULAR ANALYSES OF HIGH-AFFINITY SEROTONIN TRANSPORT

该提案概述了实现分子和免疫学 高亲和力血清素转运系统的探针，以便 分析其结构和超微结构特征。 此类研究 需要加强目前对机械作用的理解， 神经递质转运系统在神经元信号传递中， 神经病理学和药物滥用相关的神经毒性。 从这些见解中 对于某些神经系统疾病， 以及可卡因和甲基苯丙胺成瘾。 到 促进这些实验研究，高- 5-羟色胺（5-HT）的亲合转运已经通过基因转移在 我们的实验室 这些模型是两个克隆细胞系， 将人DNA转染到小鼠L-M成纤维细胞中。 两种细胞系 表现出高亲和力的5-HT转运系统， 药理学和动力学特征符合已知的那些 高亲和力5-HT转运系统;未转染的L-M细胞不 表现出这样的5-HT摄取特性。 据推测，观察到的运输 两种转染细胞中的表型是由于人5- HT转运蛋白基因。 此外，5-HT转运的膜存在 这些细胞中的系统对于细胞宿主是外源性的， 小鼠宿主在体内的免疫应答（而成纤维细胞抗原 的转染子细胞将逃避这种应答）。 因此这些 转染子模型极大地促进了实验策略，以：（1） 系统地描绘编码高亲和力的人类基因 通过表达克隆从转染基因组中获得5-羟色胺转运蛋白; 和（2）通过以下方法开发对该转运蛋白特异的单克隆抗体： 使用完整的转染细胞作为杂交瘤发育的免疫原， 筛选 分析克隆的基因，以及其相应的 cDNA，将首次产生基因组织和 5-HT转运蛋白的主要序列。 该蛋白质基因可以应用于 致突变研究，旨在确定关键的结构域， 运输功能。 鉴定的抗体将用于组织学检查 并从超微结构上检查高亲和力5-HT转运的位置 在CNS的明确的多巴胺能神经回路内的系统。

This proposal outlines approaches to attain molecular and immunological probes for the high-affinity serotonin transport system, in order to analyze its structural and ultrastructural features. Such studies are needed to enhance current understanding of the mechanistic role(s) of neurotransmitter transport systems in neuronal signal transmission, neuropathology and drug abuse-related neurotoxicity. From these insights may come better diagnoses and treatments for certain neurological disorders, as well as for cocaine- and methamphetamine-addiction. To facilitate these experimental studies, novel cellular models of high- affinity serotonin (5-HT) transport have been developed by gene transfer in our laboratory. These models are two clonal cell lines derived from transfection of human DNA into mouse L-M fibroblasts. Both cell lines exhibit high-affinity 5-HT transport systems with physiological, pharmacological and kinetic characteristics that conform to those of known high-affinity 5-HT transport systems; untransfected L-M cells do not manifest such 5-HT uptake properties. Presumably, the observed transport phenotypes in both transfectant cells are due to expression of the human 5- HT transporter gene. Further, the membrane presence of the 5-HT transport systems in these cells are exogenous to the cell host and likely to elicit an immune response by a mouse host in vivo (while the fibroblastic antigens of the transfectant cells will escape such response). Thus, these transfectant models greatly facilitate experimental strategies to: (1) systematically delineate the human gene(s) encoding the high-affinity serotonin transporter from the transfectant genomes by expression cloning; and (2) develop monoclonal antibodies specific for this transporter by using intact transfectant cells as immunogens for hybridoma development and screening. Analysis of the cloned gene, as well as its corresponding cDNAs, will yield for the first time both the gene organization and the primary sequence of the 5-HT transporter. The protein gene can be applied to mutagenesis studies aimed at identifying structural domains critical to transport function. Identified antibodies will be used to histologically and ultrastructurally examine the location of high-affinity 5-HT transport systems within well-defined serotonergic neurocircuitries of the CNS.