DOPAMINE TRANSPORTER I--STRUCTURE/FUNCTION STUDIES

多巴胺转运蛋白（DAT）已被确定为主要的大脑 受体部位与奖赏和欣快特性最相关 可卡因。对快速服用可卡因的欣快反应可能是 比那些以较慢的速度 局在以前的财政年度，本分支的调查人员发现， 蛋白激酶C（PKC）激活剂调节多巴胺转运， 瞬时表达COS细胞（Eur. J. Pharmacol.，268，115-119）。 在 本财年，我们已经开始探索直接 DAT的磷酸化可能会产生 这可能会对快速和慢速的 运输机占用率 我们复制了PKC激活剂在 稳定表达DAT的LLC-PK 1细胞，并使用特异性抗DAT 在以前的财政年度开发的抗血清，用于免疫沉淀磷酸化DAT， 纹状体切片和稳定转染的细胞制备物。 初始 有证据表明，PKC激活确实会增强DAT水平， 磷酸化，从而增加了磷酸化作为一种 多巴胺能系统快速适应的候选机制 与可卡因引起的欣快感有关

The dopamine transporter (DAT) has been identified as the principal brain receptor site best correlated with the rewarding and euphoric properties of cocaine. Euphoric responses to rapid administration of cocaine can be much more prominent than those that follow slower rates of administration. In previous FYs, investigators in this Branch have found that activators of protein kinase C (PKC) modulate dopamine transport in transiently-expressing COS cells (Eur. J. Pharmacol., 268, 115-119). In this FY, we have begun to explore the extent to which direct phosphorylation of the DAT may produce the sorts of acute adaptations that could yield differing responses to rapid and slow rates of transporter occupancy. We have replicated effects of PKC activators in LLC-PK1 cells that stably express DAT, and used specific anti-DAT antisera developed in previous FYs to immunoprecipitate phosphor-DAT from striatal slice and stably transfected cell preparations. Initial evidence suggests that PKC activation does enhance levels of DAT phosphorylation, and thus increases evidence for phosphorylation as a candidate mechanism for rapid adaptations in dopaminergic systems relevant to cocaine-induced euphoria.