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：钠同向转运蛋白（NSS）家族包括血清素、多巴胺和去甲肾上腺素的转运蛋白，它们是抗抑郁药、哌醋甲酯（利他林）和广泛滥用的精神兴奋剂可卡因和安非他明的靶标。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点。 NSS 受到底物、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 功能的分子机制是充分了解 NSS 调节以设计改进的治疗干预手段的必要基础。虽然人类 NSS 蛋白的纯化和结晶尚未实现，但原核同源物已被证明是了解 NSS 分子细节机制的强大模型系统。 LeuT 是一种钠依赖性氨基酸转运蛋白，已在晶体学上以不同的构象捕获。这些数据极大地增进了我们对 NSS 传输机制的理解。然而，这些 LeuT 的静态“快照”未能揭示对于理解 NSS 调控至关重要的门控机制的动态方面。为了解决这个缺点，我们开发了单分子福斯特共振能量转移（smFRET）方法，使我们能够直接实时测量LeuT的构象动力学。在我们发表的工作中，我们研究了 LeuT 细胞内表面的底物和抑制剂依赖性门控动力学调节。这些数据提供了关于运输机制的关键假设，我们现在的目标是通过首次对 LeuT 细胞外表面的动力学进行成像来测试和探索。这些研究将使我们能够更深入地了解细胞外表面构象事件的作用，底物和抑制剂从细胞外表面进入转运蛋白，以及这些事件与在调节底物释放的细胞内表面观察到的动态之间的关系。我建议描述 LeuT 细胞内和细胞外动力学之间的关系及其配体的调节。由于 H+ 反向转运与运输循环有关，我将定量描述 H+ 与 LeuT 结合对运输动力学的影响。此外，我将检查 Na+ 梯度对门控的调节，这是运输的生理驱动因素。这些目标的完成将有助于阐明 NSS 转运机制，并有可能为下游筛选和/或设计针对 NSS 的新型疗法提供信息。此外，该提案中开发的单分子方法将有助于建立一个平台，用于在细胞膜上原位对其他膜蛋白（包括人类 NSS）进行单分子成像。