COCAINE RECEPTOR--BIOCHEMICAL AND MOLECULAR STUDIES

因为多巴胺转运体是精神兴奋剂的关键靶点 药物，这个项目的一个目标是描述多巴胺转运蛋白 和其他运输工具。 这种蛋白质的特性， 药物受体，作为多巴胺能神经元的重要实体， 理解其功能至关重要。 我们以前已经证明多巴胺转运蛋白存在于多种细胞中， 后期修改的形式。 例如分子量 在丘脑核中的转运蛋白的含量比 在纹状体。 我们发现有证据表明 在发育和衰老过程中的翻译后变异， 多巴胺转运蛋白的分子量在此期间发生变化。 这种变化很大一部分是由于差异造成的。 糖基化。 其他蛋白质也显示出与发育相关 糖基化的变化。 因为有证据表明糖基化 是功能蛋白质的重要组成部分，这些发现与 转运体的功能方面的老化。 我们之前首次发现，多巴胺的再摄取是 被一氧化氮抑制 这似乎是一个重要的问题，因为 一氧化氮可能是一种跨突触调节剂， 从突触后元件到突触前位点来改变功能。 因为我们知道皮层神经元的突触是 与中脑的多巴胺能突触并排， 研究谷氨酸摄取是否会受到硝酸盐的影响。 环氧 事实上，我们确实发现它被一氧化氮抑制了。 这 有着重要的意义 例如，如果一氧化氮是 兴奋性氨基酸受体介导的毒性循环，然后抑制 重摄取将增强兴奋性氨基酸的毒性。 是否 这是否是一种在体内发生的相关机制 还有待观察。 我们目前正在调查 这些机制在体内起作用。 另一个项目是阐明多巴胺的结构 通过肽图谱分析转运蛋白。 在此过程中， 转运蛋白用可卡因的光亲和类似物标记， 用酶分解蛋白质，然后测量碎片， 对于分子量，光亲和标记物的含量，和 糖基化含量 这些结果表明， 基于GBR 12909的名为DEEP的文件似乎与第一个 两个跨膜螺旋，而RTI-82，一种基于 可卡因标记跨膜结构域4和12之间的蛋白质。 这些结果将最终帮助我们理解 不同转运抑制剂结合的位点。 总的来说，我们继续在了解 多巴胺转运蛋白的性质。 我们目前正专注于 对转运蛋白磷酸化的影响。

Because the dopamine transporter is a key target for psychostimulant drugs, a goal of this project is to characterize the dopamine transporter and other transporters. The characterization of this protein both as a drug receptor and as an important entity of the dopaminergic neuron is essential in understanding its function. We have previously shown that the dopamine transporter exists in multiple post-translationally modified forms. For example, the molecular weight of the transporter in the nucleus accumbens is somewhat higher than that in the striatum. We have found that there is evidence for post-translational variations during development and aging since the molecular weight of the dopamine transporter changes during this time. It appears that a significant part of this change is due to differences in glycosylation. Other proteins have also shown developmentally related changes in glycosylation. As there is evidence that the glycosylation is an important part of the functioning protein, these findings bear on the functional aspect of the transporter over aging. We previously found, for the first time, that dopamine reuptake is inhibited by nitric oxide. This appears to be an important issue because nitric oxide may be a transsynaptic regulator and may be able to diffuse from postsynaptic elements to presynaptic sites to alter function. Because it is known that glutamatergic synapses from cortical neurons are side by side with dopaminergic synapses from the midbrain, we investigated whether or not glutamate uptake would be affected by nitric oxide. In fact, we did find that it was inhibited by nitric oxide. This has important implications. For example, if nitric oxide is part of the excitatory amino acid receptor mediated toxic cycle, then inhibition of reuptake would enhance the toxicity of excitatory amino acids. Whether or not this turns out to be a relevant mechanism that occurs in vivo remains to be seen. We are currently investigating as to whether or not these mechanisms operate in vivo. Another project is the elucidation of the structure of the dopamine transporter protein by peptide mapping. In this procedure, the transporter is labeled with a photoaffinity analog of cocaine, treated with enzymes that break the protein and the fragments are then measured for molecular weight, content of the photoaffinity label, and glycosylation content. These results suggest that a photoaffinity label called DEEP which is based on GBR 12909 appears to bind within the first two transmembrane helices while RTI-82, a photoaffinity label based on cocaine, labels the protein between transmembrane domains 4 and 12. These results will ultimately help us understand the precise molecular sites where the different transport inhibitors bind. Overall, we continue to make substantial progress in understanding the nature of the dopamine transporter protein. We are currently focusing on the phosphorylation of the transporter protein.