Phosphorylation and Regulation of Dopamine Transporters

描述（由申请人提供）：多巴胺转运体（DAT）是负责从突触空间清除细胞外多巴胺的主要系统。因此，它在终止突触传递和调节可用于结合突触前和突触后多巴胺受体的多巴胺浓度方面发挥关键作用。DATs经历蛋白激酶C介导的磷酸化，并显示多巴胺转运的减少，这表明DATs经历磷酸化诱导的功能调节。这将为神经元提供一种精细的时间和空间控制细胞外多巴胺浓度的机制，以及随后的下游多巴胺能神经活动。因此，DAT磷酸化有可能深刻影响正常的多巴胺能神经生理，并可能与多巴胺能神经的机制有关。可卡因、安非他命和冰毒等精神兴奋剂的产生或滥用目前的证据表明，pkc介导的DAT调控是通过调节细胞内转运和控制质膜上DAT拷贝数发生的，但DAT磷酸化和转运之间的明确关系尚未建立。本研究全面表征了DAT磷酸化特性，明确了DAT磷酸化与功能调控之间的关系。本研究的长期目标是了解正常神经生理条件下DAT磷酸化的生理意义，以及与药物滥用的关系。为实现这些目标而设计的具体目标是：1。使用质谱法鉴定天然和表达的dat上pkc刺激磷酸化的位点。2. 构建磷酸化位点变为非磷酸盐受体的突变体，并检查其细胞内转运。3. 表征底物和精神兴奋剂如甲基苯丙胺和可卡因影响DAT磷酸化的能力。4. 确定负责DAT磷酸化生理控制的内源性途径。

DESCRIPTION (provided by applicant): The dopamine transporter (DAT) is the primary system responsible for clearance of extracellular dopamine from the synaptic space. As such, it performs a key role in terminating synaptic transmission and in regulating the concentration of dopamine available for binding to pre- and post-synaptic dopamine receptors. DATs undergo protein kinase C mediated phosphorylation and display concomitant reductions in dopamine transport, suggesting that DATs undergo phosphorylation-induced functional regulation. This would provide the neuron with a mechanism for fine temporal and spatial control of extracellular dopamine concentrations, and subsequent downstream dopaminergic neural activity. DAT phosphorylation therefore has the potential to profoundly influence normal dopaminergic neurophysiology, and may be related to mechanisms of dopaminergic neurode.qeneration or abuse of psychostimulants such as cocaine, amphetamine, and methamphetamine. Current evidence suggests that PKC-mediated regulation of DAT occurs by modulation of intracellular traffickinq and control of DAT copy numbers at the plasma membrane, but a definitive relationship between DAT phosphorylation and trafficking has not been established. This study proposes te thoroughly characterize DAT phosphorylation properties and define the relationship between DAT phosphorylation and functional regulation. The long term goal of this research is to understand the physiological significance of DAT phosphorylation under conditions of normal neurophysiology and with respect to involvement with drug abuse. The specific aims designed to achieve these goals are: 1. Identify sites of PKC-stimulated phosphorylation on native and expressed DATs using mass spectrometry. 2. Construct mutants with phosphorylation sites changed to non-phosphate acceptors, and examine their intracellu[ar trafficking. 3. Characterize the ability of substrates and psychostimulants such as methamphetamine and cocaine to affect DAT phosphorylation. 4. Identify the endogenous pathways responsible for physiological control of DAT phosphorylation.