Amphetamine Acts as a Molecular Stent in the Dopamine Transporter

描述(由申请人提供)：本申请涉及广泛的挑战领域(15)翻译科学和具体的挑战主题，15-MH-107：精神障碍药物发现的目标。苯丙胺在多巴胺转运蛋白中充当分子支架通过引入与苯丙胺和多巴胺转运蛋白相关的术语‘分子支架’，我们打算将一种新的思路引入治疗精神障碍药物发现的一般文献中。我们试图通过这个术语来框架另一种分子解释苯丙胺及其近亲甲基苯丙胺可能起作用的方法。我们知道AMPH和冰毒作用于人类多巴胺转运蛋白(HDAT)，这是这些药物的主要分子靶点。多巴胺转运体对奖赏通路中的多巴胺能信号至关重要，因此是药物滥用和成瘾的核心。我们的长期目标是研究药物滥用和成瘾的分子机制，作为药物发现的基础，我们提出了一个新的概念，新的实验证据表明，苯丙胺寄宿在转运体中，打开了一条不寻常的离子通道，由此产生的电流使多巴胺能神经元去极化，增加它们的兴奋性，并有助于解释对这些成瘾药物的反应。我们建议在一个集中的、人工的、为期两年的项目中，利用同时的放射性标记摄取和双微电极电压钳技术，广泛地研究表达HDAT的非洲爪哇卵母细胞对DA、AMPH和冰毒的摄取和诱导电流。在饱和浓度下，AMPH和冰毒比DA产生更大的电流，一旦接触这些药物，基线保持电流就会持续地使膜电位朝着去极化方向移动，这是长时间的缓冲灌流不能洗掉的。基线漂移的幅度取决于药物的浓度、暴露时间和化学结构，其中安非他明比冰毒更有效。安非他明或冰毒暴露也减少了随后DA诱导的电流，这表明暴露于安非他明或冰毒后，活跃转运体的数量受到了损害。可卡因类似物HDAT抑制剂RTI-55消除了整个去极化电流并恢复了原始基线，这表明AMPH或METH的结合并不能从膜上移除HDAT；相反，药物将转运体稳定在配体门控的通道样状态，导致通过我们描述为分子支架的转运体的泄漏电流。高正膜电位的应用只能将Amph从转运体上部分去除，这意味着Amph结合紧密，支架在药物去除后持续很长时间。通过安非他明或冰毒暴露的转运体的结构性泄漏电流可能对于理解药物成瘾的分子机制至关重要：在这个模型中，多巴胺能神经元因泄漏而去极化，因此变得更兴奋，更有可能释放递质。我们认为，AMPH或冰毒在HDAT内形成分子支架是这些药物停药后持续作用的一个重要特征。 公共卫生相关性：《苯丙胺在多巴胺转运体中充当分子支架》提案针对苯丙胺成瘾的具体问题，并介绍了用于吸毒者戒毒的药物开发策略。中心假说基于的数据表明，苯丙胺滞留在多巴胺转运体中，这为Na+离子引入了一条途径，持续地使多巴胺能神经元去极化，不适当地释放多巴胺。药物开发依赖于苯丙胺和多巴胺之间的化学相似性，苯丙胺引入支架，多巴胺不引入支架。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (15) Translational Science and specific Challenge Topic, 15-MH-107: Targets for Drug Discovery for Mental Disorders. Amphetamine acts as a Molecular Stent in the Dopamine Transporter By introducing the term 'molecular stent' in connection with amphetamine and the dopamine transporter, we are intending to introduce a new line of thinking into the general body of literature on drug discovery for mental disorders. We attempt through this term to frame another approach to the molecular explanation of how amphetamine and its close relative, methamphetamine, might work. We know that AMPH and METH act at human dopamine transporter (hDAT), which is the primary molecular target for these drugs. Dopamine transporters are critical to dopaminergic signaling in reward pathways, and therefore are central to substance abuse and addiction. With the long-term objective to study the molecular mechanisms underlying drug abuse and addiction as a basis for drug discovery, we propose a new concept suggested by new experimental evidence that amphetamines lodge in transporter and open a pathway for ions that is not ordinarily there, and that the currents thus generated depolarize dopaminergic neurons, increase their excitability, and help explain the response to these addictive drugs. We propose to study extensively, in a concentrated, well manned, two-year project, DA, AMPH, and METH uptake and induced currents in hDAT-expressing Xenopus oocytes using simultaneous radio-labeled uptake and two-microelectrode voltage-clamp techniques. At saturating concentrations, AMPH and METH induce larger currents than DA, and, once exposed to these drugs, a baseline holding current persistently shifted the membrane potential in the depolarizing direction, which a long period of buffer perfusion could not wash out. The magnitude of baseline shifts depends on the concentration, the duration of exposure, and the chemical structure of the drugs, with AMPH more potent than METH. AMPH or METH exposure also reduced subsequent DA-induced currents, suggesting that the number of active transporters is compromised after exposure to AMPH or METH. RTI-55, a cocaine analogue hDAT inhibitor, eliminated the entire depolarizing current and restored the original baseline, suggesting the binding of AMPH or METH does not remove hDAT from the membrane; rather, the transporter is stabilized by the drugs to a ligand-gated, channel-like state causing a leak current through the transporter we describe as a molecular stent. An application of high positive membrane potentials only partially removed AMPH from the transporter, implying AMPH binds tightly and the stent persists long after the drug is removed. The constitutive leak current through the AMPH- or METH-exposed transporter is likely to be crucial to understanding the molecular mechanism of drug addiction: in this model, dopaminergic neurons would be depolarized by the leak, therefore becoming more excitable and more likely to release transmitter. We propose that the formation of a molecular stent by AMPH or METH within hDAT is an important feature of the persistent effects of these drugs after withdrawal. PUBLIC HEALTH RELEVANCE: The proposal 'Amphetamine acts as a Molecular Stent in the Dopamine Transporter' addresses the specific problem of amphetamine addiction, and introduces drug- development strategies for detoxification of drug abusers. The central hypothesis rests on data suggesting that amphetamines become lodged in the dopamine transporter, which introduces a pathway for Na+ ions that persistently depolarize dopaminergic neurons, releasing dopamine inappropriately. Drug development relies on chemical similarities between amphetamine, which introduces a stent, and dopamine, which does not.