MOLECULAR STUDIES OF CATECHOLAMINE TRANSPORTERS

钠依赖儿茶酚胺转运体有很大影响 中枢多巴胺能和去甲肾上腺素能神经元的净突触输出 系统，是滥用兴奋剂的主要行动场所 比如可卡因和安非他明。我们的实验室分离出了一名人类 编码具有底物专一性的转运活性的cDNA和 去甲肾上腺素转运体的药理学特性，包括 对抗抑郁药物和精神运动兴奋剂都敏感。这个 在本申请中提出的研究使用分子生物学 表征细胞的结构、功能和调控的技术 儿茶酚胺转运蛋白。这个基因家族的其他成员 将通过序列同源性和聚合酶链式反应从cDNA文库中分离得到 (聚合酶链式反应)基于序列的策略 克隆的钠依赖去甲肾上腺素转运体。两位候选人已经 已被鉴定：去甲肾上腺素的一种潜在的神经胶质形式 转运体mRNA和编码具有 中枢神经系统的分布与多巴胺携带者一致。 该方案中的初步研究旨在表征底物 各转运蛋白基因的特异性和药理敏感性 通过将其导入多种细胞类型来表达。一组 将针对预测的决定因素提高特定的抗血清 蛋白质来评估每个基因产物的解剖分布，以 确认从序列分析预测的结构模型，并解决 这些蛋白质的细胞生物学的关键方面。这些抗血清将 与其他分子技术结合使用来研究 运输活性和转运蛋白基因表达的调节。 这项提案的另一个目标是建设和 鉴定嵌合和突变转运蛋白的特性 功能结构域包括底物的结合位点和 像可卡因这样的药理物质。基因克隆的可得性 编码儿茶酚胺载体将允许精确的结构功能 在没有囊泡储存的转基因细胞中进行的研究 不受其他运输工具的混杂影响 小路。儿茶酚胺转运蛋白的详细特性 以上所获得的结构、细胞生理和调节 研究应该确定刺激性药物的初始作用部位和 为今后研究黄曲霉毒素的作用机制奠定坚实的基础 上瘾。

The sodium dependent catecholamine transporters have a major influence on the net synaptic output of central dopaminergic and noradrenergic systems and are the primary site of action for stimulant drugs of abuse such as cocaine and amphetamine. Our laboratory has isolated a human cDNA encoding a transport activity with the substrate-specificity and pharmacologic properties of a norepinephrine transporter, including sensitivity to both antidepressants and psychomotor stimulants. The investigations proposed in this application use molecular biologic techniques to characterize the structure, function and regulation of catecholamine transport proteins. Additional members of this gene family will be isolated from cDNA libraries using sequence homology and PCR (polymerase chain reaction) strategies based on the sequence of the cloned sodium-dependent norepinephrine transporter. Two candidates have already been identified: a potential glial form of the norepinephrine transporter mRNA and an mRNA encoding a related transporter with a distribution in the CNS consistent with a dopamine carrier. Initial studies in this proposal aim at characterizing the substrate specificity and pharmacologic sensitivity of each transporter cDNA expressed by transfection into a variety of cell types. A panel of specific antisera will be raised against determinants of the predicted proteins to evaluate the anatomic distribution of each gene product, to confirm structural models predicted from sequence analysis and to address key aspects of the cell biology of these proteins. These antisera will be used in conjunction with other molecular techniques to study the regulation of both transport activity and transporter gene expression. An additional goal of this proposal is the construction and characterization of chimeric and mutant transporters to identify functional domains including binding sites for substrates and pharmacologic agents such as cocaine. The availability of cDNA clones encoding catecholamine carriers will allow precise structure-function studies to be undertaken in transfected cells devoid of vesicular storage Compartments and free from the confounding influences of other transport pathways. The detailed characterization of catecholamine transporter structure, cellular physiology and regulation obtained by the above studies should define the initial site of action of stimulant drugs and provide a strong foundation for future studies of the mechanisms of addiction.