THE DOPAMINE TRANSPORTER - IMMUNOCHEMICAL CHARACTERIZATION AND LOCALIZATION IN BRAIN
THE DOPAMINE TRANSPORTER - IMMUNOCHEMICAL CHARACTERIZATION AND LOCALIZATION IN BRAIN
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DOI：
10.1523/jneurosci.15-03-01714.1995
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发表时间：
1995-03-01
影响因子：
5.3
通讯作者：
LEVEY, AI
中科院分区：
文献类型：
--
作者：
CILIAX, BJ;HEILMAN, C;LEVEY, AI
用免疫印迹分析、免疫沉淀和免疫细胞化学等方法对多巴胺转运蛋白(DAT)进行了免疫印迹分析、免疫沉淀和免疫细胞化学表征，并在光镜水平上对大鼠脑内转运蛋白进行免疫定位。针对N末端、第二细胞外环和C末端的抗体是由含有这些区域的氨基酸序列的融合蛋白产生的。免疫印迹分析表明，N末端和环抗体对表达的克隆DAT具有特异性，可在大鼠和人纹状体膜上识别转运蛋白，对过量同源融合蛋白的预吸收敏感。免疫沉淀研究表明，抗DAT抗血清以浓度依赖的方式识别溶解的、放射性标记的DAT蛋白。这些抗体的DAT免疫细胞化学对融合蛋白的预吸收以及多巴胺能中纹状体和中皮质边缘通路的损伤也很敏感。DAT的区域分布与腹侧中脑、内侧前脑束、背侧和腹侧纹状体的多巴胺能神经支配相一致。然而，DAT和酪氨酸羟化酶的免疫细胞化学分布之间存在明显的不匹配，表明多巴胺能系统是异质性的，可能使用独立的机制来调节大脑中的多巴胺水平。特异性DAT抗体的产生将进一步表征DAT蛋白的细胞和亚细胞定位，以及神经和精神疾病中的多巴胺能回路。
Antibodies specific for the dopamine transporter (DAT) were developed and characterized by immunoblot analysis, immunoprecipitation, and immunocytochemistry, and used for immunolocalization of transporter protein in rat brain at the light microscopic level. Antibodies targeting the N-terminus, the second extracellular loop, and the C-terminus were generated from fusion proteins containing amino acid sequences from these respective regions. Immunoblot analysis demonstrated that N-terminus and loop antibodies were specific for expressed cloned DAT, recognized transporter protein in rat and human striatal membranes, and were sensitive to preabsorption with excess homologous fusion protein. Immunoprecipitation studies demonstrated that anti-DAT antisera recognized solubilized, radiolabeled DAT protein in a concentration-dependent manner. DAT immunocytochemistry with these antibodies were also sensitive to preabsorption with fusion protein and to lesions of dopaminergic mesostriatal and mesocorticolimbic pathways. Regional distribution of DAT coincided with established dopaminergic innervation of several regions, including ventral mesencephalon, medial forebrain bundle, and dorsal and ventral striatum. However, certain mismatches between immunocytochemical distributions of DAT and tyrosine hydroxylase were apparent, indicating that dopaminergic systems are heterogeneous and may use independent mechanisms for the regulation of dopamine levels in brain. The generation of specific DAT antibodies will permit further characterization of the cellular and subcellular localization of DAT protein, and of dopaminergic circuits in neurological and psychiatric disorders.