Phosphorylation and Regulation of Dopamine Transporters

描述（由申请人提供）：多巴胺转运蛋白（DAT）是负责从突触间隙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。DAT经历蛋白激酶C介导的磷酸化，并显示伴随的多巴胺转运减少，表明DAT经历磷酸化诱导的功能调节。这将为神经元提供精细的时间和空间控制细胞外多巴胺浓度以及随后的下游多巴胺能神经活性的机制。因此，DAT磷酸化具有深刻影响正常多巴胺能神经生理学的潜力，并且可能与多巴胺能神经变性或滥用精神兴奋剂如可卡因、苯丙胺和甲基苯丙胺的机制有关。目前的证据表明，PKC介导的DAT调节通过调节细胞内运输和控制DAT在质膜上的拷贝数而发生，但DAT磷酸化和运输之间的明确关系尚未建立。本研究旨在深入研究DAT磷酸化的特性，并明确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DAT磷酸化在正常神经生理学条件下的生理学意义以及与药物滥用的关系。旨在实现这些目标的具体目标是：1。使用质谱法鉴定天然和表达DAT上PKC刺激的磷酸化位点。2.构建磷酸化位点变为非磷酸受体的突变体，并检测其胞内转运。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磷酸化的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.