MOLECULAR STUDIES OF CATECHOLAMINE TRANSPORTERS

钠依赖性儿茶酚胺转运蛋白对 对中枢多巴胺能和去甲肾上腺素能神经元的突触净输出的影响 是滥用兴奋剂药物的主要作用部位 例如可卡因和安非他明。 我们的实验室分离出一个人类 编码具有底物特异性的转运活性的cDNA， 去甲肾上腺素转运蛋白的药理学特性，包括 抗抑郁药和精神兴奋剂的敏感性。 的 本申请中提出的研究使用分子生物学 技术来表征的结构，功能和调节 儿茶酚胺转运蛋白 该基因家族的其他成员 将使用序列同源性和PCR从cDNA文库中分离 （聚合酶链反应）策略的基础上的序列的 克隆的钠依赖性去甲肾上腺素转运蛋白 两名候选人 已经被确定：去甲肾上腺素的一种潜在的神经胶质形式 转运蛋白mRNA和编码相关转运蛋白的mRNA， 在中枢神经系统中的分布与多巴胺载体一致。 该提案中的初步研究旨在表征底物 每种转运蛋白cDNA的特异性和药理学敏感性 通过转染到多种细胞类型中表达。 一组 特异性抗血清将针对预测的 蛋白质，以评估每个基因产物的解剖分布， 确认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，以研究 转运活性和转运蛋白基因表达的调节。 该提案的另一个目标是建设和 嵌合和突变转运蛋白的表征以鉴定 功能结构域，包括底物的结合位点， 药物如可卡因。 cDNA克隆的可用性 编码儿茶酚胺载体将允许精确的结构-功能 在缺乏囊泡储存的转染细胞中进行的研究 舒适且不受其他运输的混杂影响 途径。 儿茶酚胺转运蛋白的详细特性 结构、细胞生理学和调节， 研究应确定兴奋剂药物的初始作用部位， 为今后的机制研究奠定了坚实的基础， 成瘾

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.