smFRET Investigation of Gating in a Neurotransmitter Transporter Homolog

神经递质转运蛋白同系物门控的 smFRET 研究

• 批准号: 8722381
• 负责人: Rachel Ann Kolster
• 金额: $ 4.27万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8722381
• 关键词: Address; Amino Acid Transporter; Amphetamines; Antidepressive Agents; Anxiety Disorders; Binding; Biological Models; Cell membrane; Chloride Ion; Cocaine; Complex; Crystallization; Data; Detergents; Dopamine; Energy Transfer; Epilepsy; Equilibrium; Event; Face; Family; Foundations; Glutamates; Glycine; Homologous Gene; Human; Image; Imaging Techniques; In Situ; Investigation; Ion Cotransport; Ions; Kinetics; Label; Ligands; Measures; Membrane Proteins; Membrane Transport Proteins; Mental disorders; Methods; Methylphenidate; Molecular; Molecular Conformation; Neurotransmitters; Norepinephrine; Physiological; Post-Translational Protein Processing; Proteins; Protons; Psychiatric therapeutic procedure; Publishing; Regulation; Research; Ritalin; Role; Schizophrenia; Serine; Serotonin; Site; Sodium; Testing; Therapeutic; Therapeutic Intervention; Time; Training; Work; antiport; career; design; extracellular; falls; gamma-Aminobutyric Acid; improved; inhibitor/antagonist; new therapeutic target; proteoliposomes; protonation; public health relevance; reconstitution; serotonin transporter; single molecule; stimulant abuse; symporter

DESCRIPTION (provided by applicant): The neurotransmitter: sodium symporter (NSS) family includes the transporters for serotonin, dopamine, and norepinephrine, which are targeted by antidepressants, methylphenidate (Ritalin), and the widely abused psychostimulants cocaine and amphetamine. In addition, NSS for GABA and glycine are promising targets for the treatment of epilepsy and anxiety disorders or schizophrenia, respectively. NSS are regulated by a complex interplay of substrates, ions, post-translational modifications and interacting proteins. Elucidating the molecular mechanism of NSS function is a necessary foundation for understanding NSS regulation in sufficient detail to design improved means of therapeutic intervention. While the purification and crystallization of human NSS proteins has yet to be achieved, prokaryotic homologues have proven powerful model systems for understanding the NSS mechanism in molecular detail. LeuT is a sodium-dependent amino acid transporter that has been captured crystallographically in distinct conformations. These data have greatly advanced our understanding of the NSS transport mechanism. However, these static "snapshots" of LeuT fall short of revealing dynamic aspects of the gating mechanisms that are critical to understanding NSS regulation. To address this shortcoming, we have developed single-molecule Forster resonance energy transfer (smFRET) methods that enable us to directly measure conformational dynamics in LeuT in real time. In our published work, we have investigated substrate- and inhibitor-dependent modulation of gating dynamics at the intracellular face of LeuT. These data have provided critical hypotheses about the transport mechanism, which we now aim to test and explore by imaging the dynamics at the extracellular face of LeuT for the first time. These investigations will enable us to gain a deeper understanding of the role of conformational events at the extracellular face, from which substrates and inhibitors enter the transporter, as well as the relationship between these events and the dynamics observed at the intracellular face that regulate substrate release. I propose to characterize the relationship between intracellular and extracellular dynamics in LeuT, and its modulation by ligands. As H+ antiport has been implicated in the transport cycle, I will quantitatively characterize the effect of H+ binding to LeuT on the kinetics of transport. Furthermore, I will examine the modulation of gating by a Na+ gradient, which is the physiological driver of transport. Completion of these aims will contribute to the elucidation of the NSS transport mechanism, potentially informing downstream efforts to screen and/or design novel therapeutics targeting the NSS. Furthermore, the single-molecule methods developed in this proposal will aid in the establishment of a platform for single-molecule imaging of other membrane proteins, including the human NSS, in situ on the cell membrane.

描述（由申请人提供）：神经递质：钠对孢子（NSS）家族包括羟色胺，多巴胺和去甲肾上腺素的转运蛋白，这些转运蛋白是抗抑郁药，甲基苯甲酯（Ritalin）的靶向，以及广泛滥用的心理刺激剂和半乙酰氨基胺和半乙酰胺。此外，GABA和甘氨酸的NSS分别是治疗癫痫和焦虑症或精神分裂症的有希望的靶标。 NSS受底物，离子，翻译后修饰和相互作用蛋白质的复杂相互作用的调节。阐明NSS功能的分子机制是理解NSS调节的必要基础，以详细地设计改进的治疗干预手段。尽管人类NSS蛋白的纯化和结晶尚未实现，但原核生物同源物已证明了有力的模型系统，可从分子细节中理解NSS机制。 Leut是一种依赖钠的氨基酸转运蛋白，已在晶体学上以不同的构型捕获。这些数据极大地提高了我们对NSS运输机制的理解。但是，这些Leut的静态“快照”却没有揭示出对理解NSS调节至关重要的门控机制的动态方面。为了解决这一缺点，我们开发了单分子福斯特共振能量传递（SMFRET）方法，使我们能够直接实时测量Leut中的构象动力学。在我们发表的工作中，我们研究了Leut细胞内面部的底物和抑制剂依赖性调节。这些数据提供了有关运输机制的关键假设，我们现在旨在通过首次将动力学成像在Leut的细胞外面进行测试和探索。这些研究将使我们能够更深入地了解构象事件在细胞外面上的作用，底物和抑制剂从中进入转运蛋白，以及这些事件与在调节底物释放的细胞内面部观察到的动力学之间的关系。我建议表征Leut中细胞内和细胞外动力学之间的关系，并通过配体调节。由于H+ Antiport与运输周期有关，因此我将定量地表征H+结合Leut对运输动力学的影响。此外，我将检查Na+梯度对走门的调节，Na+梯度是运输的生理驱动力。这些目标的完成将有助于阐明NSS运输机制，并有可能告知下游努力筛查和/或设计针对NSS的新型治疗方法。此外，本提案中开发的单分子方法将有助于建立一个平台，以对其他膜蛋白（包括人类NSS）的单分子成像（包括人NSS）进行原位。