Dopamine Transporter--structure/function Studies Of Tran

多巴胺转运体（DAT）是一个主要的脑受体部位，与可卡因的奖赏和欣快特性相关。MNB科学家克隆了DAT cDNA和基因，发现DAT和SERT的缺失是消除小鼠可卡因条件位置偏好所必需的。DAT是目前产生帕金森病最佳模型的每种多巴胺选择性毒素的作用所必需的。今年，对DAT结构-功能关系及其与SERT关系的分析仍在继续，进一步表征了选定氨基酸侧链在转运蛋白功能中的作用。这些研究集中在多巴胺转运蛋白中感兴趣的单域和多域氨基酸变化上，以及在今年报告的其他研究中鉴定的DAT序列的人类等位基因变体产生的氨基酸变化的表征上。研究已经鉴定了人5'和3' DAT单倍型，并致力于将常见的5'单倍型变体与体内DAT表达水平相关联。在这一年中报道的研究记录了丝氨酸和苏氨酸取代对激活或抑制PKC、MAP、MEK激酶和IP 3激酶途径的药物的DAT活性作用的惊人的大的影响。在这一年完成的研究还确定了一个大的影响，共表达的PKC依赖性PP 1抑制剂，KEPI（在本实验室确定为吗啡上调基因）的功能，共表达DAT。在这一年完成的研究还文件DAT点突变对流出的帕金森氏病产生毒素MPP+和多巴胺从细胞表达DAT及其突变体的选择性影响。这些见解应继续有助于鉴定可能在体内具有可卡因拮抗剂活性的小分子化合物的结构-功能特征、与DAT调节相关的结构-功能关系以及DAT表达水平和药理学的人类个体差异。

The dopamine transporter (DAT) has been a principal brain receptor site that has been correlated with the rewarding and euphoric properties of cocaine. MNB scientists cloned the DAT cDNA and gene, and found that deletion of both DAT and SERT are required to eliminate cocaine conditioned place preferences in mice. DAT is required for the actions of each of the current dopamine-selective toxins that produce the best models of Parkinson's disease. Analyses of DAT structure- function relationships, and their relationships with SERT, continued during this year with further characterization of the roles of selected amino acid sidechains in transporter functions. These studies have focused on single- and multiple-domaine amino acid changes of interest in the dopamine transporter and on characterization of the amino acid changes produced by human allelic variants of the DAT sequence identified in other studies reported during this year. Studies ahve identified human 5' and 3' DAT haplotypes and have worked to correlate common 5' haplotype variants with levels of DAT expression in vivo. Studies reported during this year document surprisingly large effects of serine and threonine substitutions on the DAT activity effects of drugs that activate or inhibit PKC, MAP, MEK kinase, and IP3 kinase pathways. Studies completed during this year have also identified a large effect of coexpression of the PKC-dependent PP1 inhibitor, KEPI (identified in this laboratory as a morphine-upregulated gene)on the function of coexpressed DAT. Studies completed during this year also document surprisingly-selective effects of DAT point mutations on efflux of the Parkinson's disease producing toxin MPP+ and dopamine from cells expressing DAT and its mutants. These insights should continue to help in identification of structure- function features of small molecule compounds possibly active in vivo as cocaine antagonists, structure-function relationships relevant to DAT regulation, and human individual differences in DAT levels of expression and pharmacology.