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.

项目总结/摘要 安非他明类药物作用于调节情绪和行为的转运蛋白，如多巴胺 和5-羟色胺转运蛋白，并用于治疗注意力缺陷多动障碍 （多动症）。这些药物也以甲基苯丙胺和3，4-二羟甲基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蛋白，广泛用于治疗 精神障碍，属于神经递质钠同向转运体（NSS）家族的次级活性 转运蛋白，负责从突触再摄取神经递质。安非他明， 作为较大的单胺释放剂（MRA）药物家族的一部分，作用于5-羟色胺和多巴胺转运蛋白 抑制和逆转再摄取过程，导致神经递质流出进入突触。泛滥 与神经递质的突触导致极端的刺激作用，并使这些药物高度上瘾。 外排被认为是通过两种不同的模式发生的，一种是遵循规范交替的反向运输模式， 访问模型的运输，和突发外排模式，其特征在于大，瞬时通量的基板。突发 外排不符合交替进入运输模型。因此，转发了两个新模式 解释其机制：一个同步模型，其中许多转运蛋白同时释放底物， 同步的方式，或类似通道的模式，其中单个运输商通过类似通道的方式操作。 机制，以允许大通量的基板。我们假设安非他明诱导的爆发性外排是 NSS的一种通道样机制，允许底物易位的大的瞬时速率。到 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）测定，其能够以单个转运蛋白分辨率定量转运过程。这 该测定利用了蛤壳样底物结合蛋白的工程改造， 确定单个运输工具方向的方法。我们将验证这种新方法，因为 研究运输，使用细菌NSS同系物MhsT，这将使我们能够表征 运输过程的速率分布和随机性，无法使用 基于集合的方法。我们将使该测定适用于人血清素转运蛋白（hSERT），以确定 如果已知方向的单个转运蛋白在安非他明存在下表现出瞬时的大的外排率。 我们将描述这些事件的速率、选择性和方向性，以确定它们是否确实如此， 就像一个频道。然后将利用这一点来研究其他MRA的外排特性，以确定 与特定药物表型相关。我们将使用这个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，更少上瘾的治疗方法。 此外，该平台的开发将对NSS领域以及 更广泛的二级运输领域，因为它提供了一个研究这些蛋白质的全新模式。