Methamphetamine & Amphetamine Differentially Affect Dopamine Transporter Activity

描述（由申请人提供）：甲基苯丙胺（冰毒）是现存最容易上瘾和最具神经毒性的药物之一，其社会影响正在上升。甲基安非他明影响多巴胺转运体（DAT）的分子机制（DAT是几种精神活性药物的主要分子靶点）尚不清楚。重要的是，由于甲基苯丙胺和安非他明（AMPH）之间的结构相似性，甲基苯丙胺对数据的调节通常是从表征AMPH的研究中推断出来的。因此，甲基甲醚暴露DAT的生物物理特性和潜在的分子机制实际上是未知的。甲基苯丙胺主要通过产生细胞外纹状体多巴胺（DA）的大量升高来发挥其成瘾特性。DAT是一种神经递质转运体，通过清除突触释放的DA来调节突触信号的大小和持续时间。然而，DAT也通过反向转运（外排）介导DA释放，并可在通道模式下运作，从而显著增加DA通量。甲基安非他明通过DAT介导DA外排，揭示这种外排的机制对于理解甲基安非他明成瘾和神经毒性至关重要。拟议的研究将测试甲基安非他明调节细胞外DA的假设：通过稳定DAT通道模式活动来增加DA外排，减少DA摄取，和/或以电压和磷酸化依赖的方式改变DAT细胞表面分布，这些协调的事件解释了甲基安非他明与结构同属物（如AMPH）相比具有高度成瘾性和神经毒性。我们将测试这些假设，所有这些假设都得到了有希望的初步数据的支持，具体目标如下：1)确定甲基安非他明诱导的DA外排相对于AMPH的生物物理和分子机制；2)验证甲基安非他明靶向DAT磷酸化状态来调节DA外排、底物摄取和DAT表面分布的假设；3)比较甲基安非他明诱导的和AMPH诱导的电流-底物比；4)测量甲基安非他明诱导的DAT表面迁移率作为DAT n端磷酸化的函数。我们将在中脑多巴胺能神经元和表达DAT的卵母细胞中实现这些目标，使用全细胞、细胞附着和细胞分离膜片钳与同步安培法测量DA外排；并使用荧光底物ASP+来监测dat依赖性摄取。我们预计，我们的发现将确定新的治疗策略的机制，可以预防或逆转甲基安非他明毒性/成瘾，以及为其他神经系统疾病的病因包括多巴胺能系统功能障碍提出独特的靶点。

DESCRIPTION (provided by applicant): Methamphetamine (METH) is one of the most addictive and neurotoxic drugs in existence whose societal impact is on the rise. The molecular mechanisms underlying the effects of METH on the dopamine transporter (DAT), the major molecular target of several psychoactive drugs, are poorly understood. Importantly, due to structural similarities between METH and amphetamine (AMPH), METH regulation of DAT is generally inferred from studies characterizing AMPH. Therefore, the biophysical properties and underlying molecular mechanisms of METH-exposed DAT are virtually unknown. METH primarily exerts its addictive properties by producing large elevations in extracellular striatal dopamine (DA). The DAT is a neurotransmitter transporter that regulates the magnitude and duration of synaptic signaling by clearing released DA from the synapse. However, DAT also mediates DA release via reverse transport (efflux) and can operate in a channel mode, which dramatically increases DA flux. METH mediates DA efflux via DAT, and revealing the mechanisms for this efflux is critical in understanding METH addiction and neurotoxicity. The proposed studies will test the hypotheses that METH regulates extracellular DA by: stabilizing DAT channel mode activity to increase DA efflux, decreasing DA uptake, and/or modifying DAT cell surface distribution in a voltage- and phosphorylation-dependent manner, and that these coordinated events account for the highly addictive nature and neurotoxicity of METH when compared with structural congeners, like AMPH. We will test these hypotheses, all of which are supported by promising preliminary data, with the following specific aims: 1) Determine the biophysical and molecular mechanisms underlying METH-induced DA efflux relative to AMPH, 2) Test the hypothesis that METH targets a phosphorylated state of DAT to regulate DA efflux, substrate uptake, and DAT surface distribution, 3) Compare METH-induced with AMPH-induced current-to-substrate ratios 4) Measure METH-provoked DAT surface mobility as a function of DAT N-terminal phosphorylation. We will achieve these aims in midbrain dopaminergic neurons and DAT expressing oocytes using whole-cell, cell-attached, and cell-detached patch clamp with simultaneous amperometry to measure DA efflux; and use the fluorescent substrate ASP+ to monitor DAT-dependent uptake. We anticipate that our findings will identify mechanisms for novel therapeutic strategies that may prevent or reverse METH toxicity/addiction, as well as suggest unique targets for other neurological diseases whose etiology includes dysfunction of the dopaminergic system. PUBLIC HEALTH RELEVANCE: Chronic use of methamphetamine can lead to addiction, severe neurological and psychiatric impairment, as well as pronounced neurodegeneration. Due to structural similarities regulation of dopamine transporter (DAT) is generally inferred from studies characterizing other amphetamines. Our proposed studies 1) will clarify the molecular mechanisms of methamphetamine-induced dopamine efflux via DAT, and 2) differentiate methamphetamine from its well-characterized, less neurotoxic congener, amphetamine that are valuable for identification of therapeutic strategies for the treatment of methamphetamine addiction and other neurodegenerative diseases.