Methamphetamine & Amphetamine Differentially Affect Dopamine Transporter Activity

描述（由申请人提供）：甲基苯丙胺（METH）是现有最具成瘾性和神经毒性的药物之一，其社会影响正在上升。METH对多巴胺转运体（DAT）的作用的分子机制，几种精神活性药物的主要分子靶点，知之甚少。重要的是，由于METH和安非他明（AMPH）之间的结构相似性，一般从表征AMPH的研究中推断出DAT的METH调节。因此，MET暴露DAT的生物物理特性和潜在的分子机制几乎是未知的。METH主要通过产生细胞外纹状体多巴胺（DA）的大幅升高来发挥其成瘾性。DAT是一种神经递质转运蛋白，通过清除突触释放的DA来调节突触信号传导的幅度和持续时间。然而，DAT也通过反向转运（外排）介导DA释放，并可以在通道模式下工作，这大大增加了DA通量。METH通过DAT介导DA外排，揭示这种外排的机制对于理解METH成瘾和神经毒性至关重要。拟议的研究将测试METH通过以下方式调节细胞外DA的假设：稳定DAT通道模式活性以增加DA流出，减少DA摄取和/或以电压和磷酸化依赖性方式修改DAT细胞表面分布，并且与结构同源物（如AMPH）相比，这些协调事件解释了METH的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METH诱导的DA流出相对于AMPH的生物物理和分子机制，2）检验METH靶向DAT的磷酸化状态以调节DA流出、底物摄取和DAT表面分布的假设，3）比较MET诱导的与AMPH诱导的电流与底物的比率4）测量作为DAT N-末端磷酸化的函数的MET引起的DAT表面迁移率。我们将在中脑多巴胺能神经元和DAT表达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，同时安培法测量DA流出，并使用荧光底物ASP+监测DAT依赖性摄取。我们预计，我们的研究结果将确定新的治疗策略，可以防止或逆转METH毒性/成瘾的机制，以及建议其他神经系统疾病的病因包括多巴胺能系统功能障碍的独特目标。 公共卫生相关性：长期使用甲基苯丙胺可导致成瘾、严重的神经和精神损害以及明显的神经变性。由于结构上的相似性，多巴胺转运蛋白（DAT）的调节通常是从表征其他安非他明的研究中推断出来的。我们提出的研究1）将阐明甲基苯丙胺通过DAT诱导多巴胺外排的分子机制，2）将甲基苯丙胺与其特征良好的、神经毒性较小的同类物苯丙胺区分开来，这对于确定治疗甲基苯丙胺成瘾和其他神经退行性疾病的治疗策略是有价值的。

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.