Methamphetamine & Amphetamine Differentially Affect Dopamine Transporter Activity

描述(申请人提供)：甲基苯丙胺(冰毒)是现存的最容易上瘾和最具神经毒性的毒品之一，其社会影响正在上升。冰毒对多巴胺转运体(DAT)的影响的分子机制尚不清楚，DAT是几种精神活性药物的主要分子靶标。重要的是，由于冰毒和安非他明之间的结构相似，冰毒对DAT的调节通常是从表征安非他明的研究中推断出来的。因此，冰毒暴露的DAT的生物物理性质和潜在的分子机制几乎是未知的。冰毒主要通过在细胞外纹状体多巴胺(DA)中产生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)调节DA的释放，并且可以在通道模式下工作，这显著增加了DA的流量。冰毒通过DAT介导DA外流，揭示这种外流的机制对于了解冰毒成瘾和神经毒性至关重要。拟议的研究将检验以下假说：稳定DAT通道模式的活动以增加DA外流，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，以及这些协调事件解释了与结构类似物(如AMPH)相比，METH高度成瘾的性质和神经毒性。我们将测试这些假说，所有这些假说都得到了有希望的初步数据的支持，具体目的如下：1)确定冰毒诱导的DA外流相对于Amph的生物物理和分子机制；2)测试METh靶向DAT的磷酸化状态以调节DA外流、底物摄取和DAT表面分布的假设；3)比较冰毒诱导的和AMPH诱导的电流与底物的比率4)测量冰毒诱导的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的膜片钳结合同步安培技术来测量DA的外流；并使用荧光底物ASP+来监测DAT依赖的摄取。我们预计，我们的发现将确定新的治疗策略的机制，可以预防或逆转冰毒/成瘾，并为其他神经系统疾病提供独特的靶点，这些疾病的病因包括多巴胺能系统功能障碍。

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.