Amphetamine Acts as a Molecular Stent in the Dopamine Transporter

安非他明充当多巴胺转运蛋白中的分子支架

• 批准号: 7943888
• 负责人: LOUIS J DE FELICE
• 金额: $ 50万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7943888
• 关键词: Address; Amphetamine Abuse; Amphetamine Addiction; Amphetamines; Area; Attenuated; Binding; Biotinylation; Buffers; Charge; Chemical Structure; Chemicals; Cocaine; Cognition; Data; Development; Dopamine; Dopamine Agonists; Drug Addiction; Drug Metabolic Detoxication; Drug abuse; Exhibits; Exposure to; Gated Ion Channel; Human; Human Resources; Ions; Label; Lead; Legal patent; Ligands; Literature; Mammalian Cell; Measures; Mediator of activation protein; Membrane; Membrane Potentials; Mental disorders; Methamphetamine; Microelectrodes; Midbrain structure; Modeling; Molecular; Molecular Target; Moods; Nature; Oocytes; Pathway interactions; Perfusion; Pharmaceutical Preparations; Phosphorylation; Play; Preparation; Procedures; Property; Radio; Radioactive; Regulation; Relative (related person); Rest; Rewards; Role; Signal Transduction; Stents; Structure; Substance abuse problem; Surface; Symptoms; System; Testing; Thinking; Translational Research; United States; Voltage-Clamp Technics; Western Blotting; Withdrawal; Work; Xenopus oocyte; addiction; analog; base; calmodulin-dependent protein kinase II; combinatorial; design; dopamine transporter; dopaminergic neuron; drug abuser; drug development; drug discovery; drug of abuse; functional group; inhibitor/antagonist; ligand gated channel; methamphetamine exposure; novel; novel strategies; psychostimulant; public health relevance; research study; response; uptake; voltage clamp

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.

描述（由申请人提供）：本申请涉及广泛的挑战领域 (15) 转化科学和具体挑战主题 15-MH-107：精神障碍药物发现的目标。安非他明充当多巴胺转运蛋白中的分子支架通过引入与安非他明和多巴胺转运蛋白相关的术语“分子支架”，我们打算在精神障碍药物发现的一般文献中引入一种新的思路。我们试图通过这个术语构建另一种分子方法来解释安非他明及其近亲甲基安非他明如何发挥作用。我们知道 AMPH 和 METH 作用于人类多巴胺转运蛋白 (hDAT)，而 hDAT 是这些药物的主要分子靶标。多巴胺转运蛋白对于奖赏途径中的多巴胺能信号传导至关重要，因此对于药物滥用和成瘾至关重要。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，我们提出了一个由新的实验证据表明的新概念，即安非他明驻留在转运蛋白中，并为通常不存在的离子打开一条通路，从而产生的电流使多巴胺能神经元去极化，增加其兴奋性，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、精心安排的两年项目中，使用同步放射性标记摄取和两微电极电压钳技术，广泛研究表达 hDAT 的非洲爪蟾卵母细胞中 DA、AMPH 和 METH 的摄取和感应电流。在饱和浓度下，AMPH 和 METH 诱导的电流比 DA 更大，并且一旦接触这些药物，基线保持电流就会持续使膜电位向去极化方向移动，长时间的缓冲液灌注无法将其消除。基线变化的幅度取决于药物的浓度、暴露时间和化学结构，其中 AMPH 比 METH 更有效。 AMPH 或 METH 暴露也减少了随后的 DA 诱导电流，表明暴露于 AMPH 或 METH 后活性转运蛋白的数量受到损害。 RTI-55，一种可卡因类似物 hDAT 抑制剂，消除了整个去极化电流并恢复了原始基线，这表明 AMPH 或 METH 的结合不会从膜上去除 hDAT；相反，转运蛋白被药物稳定在配体门控的通道样状态，导致通过转运蛋白的漏电流，我们将其描述为分子支架。应用高正膜电位仅部分去除转运蛋白上的 AMPH，这意味着 AMPH 紧密结合，并且在药物去除后支架仍能长期存在。通过暴露于 AMPH 或 METH 的转运蛋白的本构泄漏电流可能对于理解药物成瘾的分子机制至关重要：在该模型中，多巴胺能神经元将因泄漏而去极化，因此变得更加兴奋并且更有可能释放递质。我们认为，AMPH 或 METH 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。 公共健康相关性：“安非他明充当多巴胺转运蛋白中的分子支架”提案解决了安非他明成瘾的具体问题，并介绍了药物滥用者戒毒的药物开发策略。中心假设基于数据表明安非他明滞留在多巴胺转运蛋白中，这引入了一条 Na+ 离子通道，持续使多巴胺能神经元去极化，从而不适当地释放多巴胺。药物开发依赖于引入支架的安非他明和不引入支架的多巴胺之间的化学相似性。