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 基于苯丙胺的药物对调节情绪和行为的转运蛋白作用，例如多巴胺 和5-羟色胺转运蛋白，并可以利用治疗方法治疗注意力缺陷多动障碍 （多动症）。这些药物还以甲基苯丙胺和3,4-的形式被广泛滥用 甲基二甲基苯丙胺。 5-羟色胺和多巴胺转运蛋白，广泛针对的治疗 精神疾病，属于神经递质钠钠象征（NSS）次级活动家族 转运蛋白，并负责突触中神经递质的重新摄取。苯丙胺， 较大的单胺释放剂（MRA）药物家族的一部分，对5-羟色胺和多巴胺转运蛋白作用 为了抑制和扭转重新摄取过程，导致神经递质排出式进入突触。洪水 神经递质的突触会产生极端的刺激作用，并使这些药物高度加性。 被认为是通过两种不同的模式出现的，一种反向传输模式遵循规范更改 传输模型和爆发效率模式，其特征在于底物的大型瞬态通量。爆裂 外排不适合替代访问传输模型。因此，已经转发了两个新型号 解释其机制：一种同步模型，其中许多转运蛋白只是在A中释放底物 同步时尚或单个转运蛋白通过频道样运行的频道样模型 允许大量底物通量的机制。我们假设苯丙胺引起的爆发外排为 NSS的渠道样机制，可实现底物易位的大型瞬态速率。到 研究了这一假设，我们已经开发了单分子荧光共振能量转移 （SMFRET）实现单个转运蛋白分辨率的传输过程的测定。这 测定资本将类似蛤壳的底物结合蛋白的工程结合在一起 确定单个转运蛋白方向的方法。我们将验证这种新方法 使用细菌NSS同源物MHST研究运输，这将使我们能够表征 运输过程的速率和随机性的分布，本质上无法使用的内在变量 基于集合的方法。我们将对人类5-羟色胺转运蛋白（HSERT）进行调整以确定 如果在苯丙胺存在下具有已知取向的单个转运蛋白，则具有裸露的瞬态外排率。 我们将表征这些事件的速率，选择性和方向性，以确定它们是否确实如此 类似于频道。然后将利用这来研究其他MRA的外排特性以定义 与特定的药物表型相关。我们将使用这个新的实验范式调查 苯丙胺对NSS的作用，这可能有助于设计更有效，添加疗法更少。 此外，该平台的开发将对NSS领域以及 广泛的二级运输场，因为它提供了研究这些蛋白质的完全新颖的方式。