MOLECULAR NEUROBIOLOGY OF THE CHOLINE TRANSPORTER

该提案概述了针对克隆、表达、 和新的神经递质转运体的特征 主要关注高亲和力的胆碱转运蛋白。神经递质 运输过程已经牵涉到多起 神经疾病，包括肌萎缩侧索硬化症， 帕金森氏症和毒瘾。传送器知识 需要进一步评估分子的结构和调控。 这些和其他神经疾病的基础，以及为提供 为合理的治疗设计奠定了基础。 神经递质转运体cDNA将通过使用 一种基于聚合酶链式反应的同源克隆策略 技术转运蛋白同源物的区域表达模式将是 通过在大鼠脑内进行原位杂交评估，允许初步的 转运蛋白底物表型的预测。新的转运蛋白克隆 在功能表达后，将进行进一步的表征 哺乳动物细胞系。初步研究证实了这项计划的可行性和 这种以神经解剖学为基础的方法用于识别和 相关神经递质转运体的特征。我们将专注于 富含胆碱的组织和细胞系的研究 转运蛋白，以缩小对功能性胆碱的搜索范围 转运蛋白基因。 多克隆抗体将通过免疫兔而获得 人工合成的寡肽和融合蛋白对应的弱- 转运蛋白的保守区被识别并用于与 转运蛋白mRNA在大鼠脑内的原位分布。在一次 广泛表征抗体的特异性，抗体将被 用于定位大鼠大脑中的转运蛋白。最后，有能力 磷酸化将直接影响转运蛋白的功能 下定决心。

This proposal outlines studies directed towards the cloning, expression, and characterization of novel neurotransmitter transporters with a principal focus on the high-affinity choline transporter. Neurotransmitter transport processes have already been implicated in a number of neurological diseases, including amyotrophic lateral sclerosis, Parkinson's disease, and drug addiction. Knowledge of transporter structure and regulation is needed for further assessment of the molecular basis for these and other neurological diseases, as well as for providing a foundation for rational therapeutic design. Neurotransmitter transporter cDNAs will be identified through the use of a homologous cloning strategy employing polymerase chain reaction (PCR) technology. Regional expression patterns of transporter homologues will be assessed by in situ hybridization in rat brain to allow a preliminary prediction of transporter substrate phenotype. Novel transporter clones will undergo further characterization after functional expression in mammalian cell lines. Preliminary studies demonstrate the feasibility and power of this neuroanatomically-based approach for the identification and characterization of related neurotransmitter transporters. We will focus these studies on tissues and cell lines enriched for the choline transporter in order to narrow the search for a functional choline transporter cDNA. Polyclonal antibodies will be raised by immunization of rabbits with synthetic oligopeptides and fusion proteins corresponding to poorly- conserved regions of the transporters identified and used to compare with the in situ distribution of transporter mRNA in rat brain. After an extensive characterization of antibody specificity, antibodies will be used to localize the transporters in rat brain. Finally, the ability of phosphorylation to affect transporter function will be directly determined.