Kappa-Opioid Receptor Mediated Regulation of Dopamine Transport

描述（由申请人提供）：抑郁症和成瘾每年影响数百万人，给社会带来数不清的代价。多巴胺转运蛋白（DAT）是一种清除释放到细胞外空间的多巴胺（DA）的膜蛋白，是临床使用的抗抑郁药和几种滥用药物的靶点。?-阿片受体（KOR）在脑回路中富集，所述脑回路有助于情绪和动机并调节位于其中的DA神经元的基础活性。KOR系统的上调与抑郁症的发病机制和以可卡因和其他滥用药物戒断为特征的情绪失调有关。尽管正在努力开发用于治疗抑郁症和成瘾的口服有效的KOR配体，但这些药剂作用于影响行为和DA传递的下游效应物是未知的。KOR激动剂的焦虑和厌恶作用归因于腹侧纹状体（VST）中DA释放减少。然而，重要的是，VST中的KOR与多巴胺转运蛋白并置。我们的研究表明，KOR激活通过ERK 1/2依赖性磷酸化DAT的苏氨酸（Thr）53而增加DAT活性，并且选择性抑制VST中的ERK 1/2减弱KOR激动剂的不利影响。这些研究结果确定了一种新的机制，KOR配体调节突触前DA的传输，并表明，转运蛋白失调可能是一个潜在的KOR介导的改变情绪和影响的机制。本提案中的研究将检验以下假设：KOR激活通过ERK 1/2依赖性磷酸化和DAT调节调节来调节DA动力学和VST依赖性行为。具体目标1将通过确定KOR介导的DAT功能和VST表达的变化是否与Thr 53磷酸化相关，以及阻止KOR激活的操作是否与Thr 53磷酸化相关，来确定KOR相关DAT调节的细胞机制。激动剂诱发的VST中ERK 1/2激活和DAT磷酸化减弱了KOR介导的DAT功能变化。具体目标2将建立相关性，这一机制的调节突触前DA的传输，通过确定是否操纵，防止KOR激动剂诱发ERK 1/2激活和DAT磷酸化改变基础DA动力学。具体目标3将通过评估VST中KOR-ERK 1/2相关DAT调节的预防是否减弱KOR激动剂的厌恶和促抑郁样作用以及KOR拮抗剂的抗抑郁样作用来确定KOR-ERK 1/2相关DAT调节与KOR激动剂和拮抗剂的行为作用的生理相关性。还将评估KOR-ERK 1/2连接的DAT调节在介导KOR激动剂预防可卡因的运动刺激作用的功效中的作用。这些研究的结果将增强我们对KOR配体调节情绪和DA传递的神经基质的理解。此外，他们将提供新的见解DAT磷酸化的作用，在调节突触DA清除和行为。

DESCRIPTION (provided by applicant): Depression and addiction affect millions of individuals each year exerting untold costs on society. The dopamine transporter (DAT), a membrane protein that clears dopamine (DA) released into the extracellular space is a target of clinically used antidepressants and several drugs of abuse. ?-opioid receptors (KOR) are enriched in brain circuits that subserve mood and motivation and regulate the basal activity of DA neurons located therein. Upregulation of KOR systems has been implicated in the pathogenesis of depression and the mood dysregulation that characterizes withdrawal from cocaine and other drugs of abuse. Despite on-going efforts to develop orally effective KOR ligands for the treatment of depression and addiction, the downstream effectors upon which these agents act to affect behavior and DA transmission are unknown. The dysphoric and aversive effects of KOR agonists have been attributed to decreased DA release in the ventral striatum (VST). Importantly, however, KOR in VST are apposed to the dopamine transporter. Our studies show that KOR activation increases DAT activity through ERK1/2 dependent phosphorylation of threonine (Thr)53 of DAT and selective inhibition of ERK1/2 in the VST attenuates the aversive effects of KOR agonists. These findings identify a novel mechanism by which KOR ligands regulate presynaptic DA transmission and suggest that transporter dysregulation may be one mechanism underlying KOR-mediated alterations in mood and affect. The studies in this proposal will test the hypotheses that: KOR activation modulates DA dynamics and VST-dependent behaviors via ERK1/2-dependent phosphorylation and regulation of DAT. Specific Aim 1 will identify the cellular mechanisms of KOR-linked DAT modulation by determining whether KOR-mediated changes in DAT function and expression in the VST are associated with Thr53 phosphorylation and whether manipulations that prevent KOR-agonist evoked ERK1/2 activation and DAT phosphorylation in the VST attenuate KOR-mediated changes in DAT function. Specific Aim 2 will establish the relevance of this mechanism to the regulation of presynaptic DA transmission by determining whether manipulations that prevent KOR-agonist evoked ERK1/2 activation and DAT phosphorylation alter basal DA dynamics. Specific Aim 3 will determine the physiological relevance of KOR-ERK1/2 linked DAT modulation to the behavioral effects of KOR agonists and antagonists by assessing whether prevention of KOR-ERK1/2 linked DAT modulation in the VST attenuates the aversive and pro-depressive like effects of KOR agonists as well as the antidepressant-like effects of KOR antagonists. The role of KOR-ERK1/2 linked DAT modulation in mediating the efficacy of KOR agonists in preventing the locomotor stimulant effects of cocaine will also be assessed. Findings from these studies will enhance our understanding of the neural substrates upon which KOR ligands act to regulate mood and DA transmission. Furthermore, they will provide new insights as to the role of DAT phosphorylation in regulating synaptic DA clearance and behavior.