MOLECULAR ANALYSES OF HIGH-AFFINITY SEROTONIN TRANSPORT

这项提案概述了获得分子和免疫学 探索高亲和力的5-羟色胺转运系统，以便 分析其结构和超微结构特征。这类研究是 需要加强当前对的机械作用的理解(S) 神经信号传递中的神经递质运输系统， 神经病理学和药物滥用相关的神经毒性。从这些见解中 对某些神经系统疾病可能会有更好的诊断和治疗 疾病，以及可卡因和甲基苯丙胺成瘾。至 促进这些实验研究，新的细胞模型的高度和 亲和力5-羟色胺(5-羟色胺)转运是通过基因转移在 我们的实验室。这些模型是两个克隆细胞系衍生自 人基因导入小鼠L-M成纤维细胞。两种细胞系 展示高亲和力的5-羟色胺转运系统， 符合已知药物的药理和动力学特征 高亲和力5-羟色胺转运系统；未转染的L-M细胞不 表现出这样的5-羟色胺摄取特性。据推测，观测到的交通 两种转基因细胞的表型均源于人5-羟色胺的表达 HT转运蛋白基因。此外，膜存在的5-羟色胺转运 这些细胞中的系统是细胞宿主的外源系统，并可能诱导 体内小鼠宿主的免疫反应(当成纤维细胞抗原 的转基因细胞将逃脱这种反应)。因此，这些 转基因模型极大地促进了实验策略的实施：(1) 系统地描绘了编码高亲和力的人类基因(S) 通过表达克隆从转基因基因组中获得5-羟色胺转运体； 和(2)通过以下方法研制出针对该转运蛋白的单抗 用完整的转基因细胞作为杂交瘤细胞的免疫原 放映。对克隆的基因进行分析，以及与之对应的 CDNA，将首次同时产生基因组织和 5-羟色胺转运体的初级序列。该蛋白质基因可以应用于 诱变研究的目的是确定对 运输功能。已鉴定的抗体将用于组织学检查 并在超微结构上检测高亲和力5-羟色胺转运的位置 中枢神经系统内明确的5-羟色胺能神经回路。

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.