Investigating Amphetamine Induced Efflux Modes in Neurotransmitter Sodium Symporters Using a Single Transporter Assay

使用单一转运蛋白测定研究神经递质钠同向转运蛋白中安非他明诱导的外排模式

• 批准号: 9395248
• 负责人: GABRIEL ARTHUR FITZGERALD
• 金额: $ 4.4万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9395248
• 关键词: Active Biological Transport; Address; Amino Acids; Amphetamine Abuse; Amphetamines; Attention deficit hyperactivity disorder; Behavior; Binding Proteins; Biological Assay; Carrier Proteins; Cells; Crystallization; Dependence; Detection; Development; Encapsulated; Engineering; Event; Exhibits; Family; Floods; Fluorescence; Fluorescence Resonance Energy Transfer; Goals; Health; Heterogeneity; Homologous Gene; Human; Hydrophobicity; Investigation; Label; Liposomes; Measurement; Measures; Mediating; Mental disorders; Methamphetamine; Methods; Modality; Modeling; Molecular; Molecular Conformation; Moods; Nature; Neurotransmitters; Pharmaceutical Preparations; Phenotype; Prevalence; Process; Property; Proteins; Psychiatric therapeutic procedure; Psychotropic Drugs; Publications; Radiolabeled; Reporting; Resolution; Serotonin; Societies; Sodium; Structure; Synapses; Therapeutic; Transport Process; United States; Vesicle; base; clinically relevant; cost; design; dopamine transporter; ecstasy; in vitro activity; member; monoamine; neurotransmitter release; neurotransmitter reuptake; neurotransmitter transport; novel; novel strategies; protein transport; proteoliposomes; psychostimulant; reconstitution; reuptake; sensor; serotonin transporter; serotonin-binding protein; single molecule; single-molecule FRET; social; symporter

PROJECT SUMMARY/ABSTRACT Amphetamine-based drugs act on transport proteins that regulate mood and behavior, such as the dopamine and serotonin transporters, and are harnessed to therapeutically treat attention deficit hyperactivity disorder (ADHD). These drugs are also widely abused in the form of methamphetamine and 3,4- Methylenedioxymethamphetamine. Serotonin and dopamine transporters, widely targeted in the treatment of psychiatric disorders, belong to the neurotransmitter sodium symporter (NSS) family of secondary active transport proteins and are responsible for the reuptake of neurotransmitter from the synapse. Amphetamines, part of the larger monoamine releasing agents (MRA) drug family, act on serotonin and dopamine transporters to inhibit and reverse the reuptake process, leading to efflux of neurotransmitter into the synapse. Flooding of the synapse with neurotransmitter results in extreme stimulant effects and makes these drugs highly addictive. Efflux is thought to occur via two distinct modes, a reverse transport mode that follows the canonical alternating access model of transport, and a burst-efflux mode, characterized by large, transient fluxes of substrate. Burst efflux does not fit within the alternating access transport model. Therefore, two new models have been forwarded to explain its mechanism: a synchronous model where many transporters simultaneously release substrate in a synchronized fashion, or a channel-like model in which a single transporter operates via a channel-like mechanism to allow for large fluxes of substrate. We hypothesize that amphetamine-induced burst efflux is a channel-like mechanism of NSS’s that allows for large, transient rates of substrate translocation. To investigate this hypothesis, we have developed a single-molecule fluorescence resonance energy transfer (smFRET) assay that enables the quantification of transport processes at single transporter resolution. This assay capitalizes on the engineering of clamshell-like substrate binding proteins in combination with a novel approach for determining the orientation of single transporters. We will validate this new approach, for investigating transport, using the bacterial NSS homologue MhsT, which will enable us to characterize the distribution of rates and stochasticity of the transport process, intrinsic variables that cannot be addressed using ensemble-based methods. We will adapt this assay to the human serotonin transporter (hSERT) to determine if single transporters, of known orientation, exhibit transient, large efflux rates in the presence of amphetamines. We will characterize the rate, selectivity and directionality of these events to determine if they do, indeed, resemble a channel. This will then be leveraged to investigate the efflux properties of other MRAs to define correlations with specific drug phenotypes. We will use this new experimental paradigm to investigate amphetamine action on NSS’s, which may aid in the design of more effective, and less addictive, therapeutics. Additionally, the development of this platform will have a transformative effect on both the NSS field, as well as the wider secondary transport field, as it provides a completely novel modality of studying these proteins.

项目摘要/摘要 苯丙胺类药物作用于调节情绪和行为的转运蛋白，如多巴胺。 和5-羟色胺转运体，并被用于治疗注意力缺陷多动障碍 (ADHD)。这些药物还以甲基苯丙胺和3，4-苯丙胺的形式被广泛滥用。 亚甲二氧基甲基苯丙胺。5-羟色胺和多巴胺转运体，广泛用于治疗高血压 精神障碍，属于神经递质钠离子转运体(NSS)家族的继发性活动 转运蛋白，并负责从突触重新摄取神经递质。安非他明, 更大的单胺释放剂(MRA)药物家族的一部分，作用于5-羟色胺和多巴胺转运体 抑制和逆转重摄取过程，导致神经递质外流到突触。洪水泛滥 与神经递质的突触会产生极端的刺激效应，使这些药物高度上瘾。 外流被认为通过两种不同的模式发生，一种是遵循规范交替的反向运输模式 传输的获取模型和突发-外流模式，其特征是底物的大的瞬时通量。爆裂 外流不符合交替通道传输模型。因此，已经推出了两款新车型 为了解释它的机制：一个同步模型，其中许多转运体同时释放底物在一个 同步方式，或类似通道的模式，其中单个传送器通过类似通道的 机制，以允许大流量的基材。我们假设安非他明诱导的猝发外排是 NSS的一种类似通道的机制，允许大的、瞬时的底物转运速率。至 为了研究这一假说，我们发展了单分子荧光共振能量转移 (SmFRET)能够在单一转运体分辨率下对运输过程进行量化的分析。这 分析利用了翻盖状底物结合蛋白的工程，并结合了一种新的 确定单一传送器方向的方法。我们将验证这一新方法，以 研究转运，使用细菌NSS同源物MhsT，这将使我们能够表征 速率的分布和运输过程的随机性，这些内在变量不能用 基于系综的方法。我们将把这种分析方法应用于人类5-羟色胺转运体(HSERT)，以确定 如果单一的转运体，已知的方向，在苯丙胺存在的情况下，表现出瞬时的大的外排速率。 我们将对这些事件的速度、选择性和方向性进行表征，以确定它们是否确实如此， 就像一条水道。然后，将利用这一点来调查其他MRA的外流属性，以定义 与特定药物表型的相关性。我们将使用这一新的实验范式来研究 安非他明对NSS的作用，这可能有助于设计更有效、更不容易上瘾的治疗方法。 此外，这一平台的发展将对国家安全局领域以及 更广泛的次级运输领域，因为它为研究这些蛋白质提供了一种全新的模式。