smFRET Investigation of Gating in a Neurotransmitter Transporter Homolog

描述（由申请人提供）：神经递质：钠同向转运体（NSS）家族包括5-羟色胺、多巴胺和去甲肾上腺素的转运体，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都以这些转运体为靶点。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受底物、离子、翻译后修饰和相互作用蛋白质的复杂相互作用调节。阐明NSS功能的分子机制是充分详细地理解NSS调节以设计改进的治疗干预手段的必要基础。虽然人NSS蛋白的纯化和结晶尚未实现，但原核同源物已被证明是用于在分子细节上理解NSS机制的强大模型系统。LeuT是一种钠依赖性氨基酸转运蛋白，其在不同的构象中被晶体学捕获。这些数据极大地推进了我们对NSS输运机制的理解。然而，这些静态的“快照”的LeuT不足以揭示动态方面的门控机制，这是至关重要的理解NSS监管。为了解决这个缺点，我们已经开发了单分子福斯特共振能量转移（smFRET）的方法，使我们能够直接测量构象动态LeuT在真实的时间。在我们发表的工作中，我们已经研究了底物和底物依赖性的调制门控动力学在细胞内面对LeuT。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成像LeuT细胞外表面的动态来测试和探索。这些调查将使我们能够获得更深入的了解构象事件的作用在细胞外的脸，底物和抑制剂进入转运蛋白，以及这些事件之间的关系和观察到的动态在细胞内的脸，调节底物释放。我建议LeuT的细胞内和细胞外动力学之间的关系，其调制配体的特点。由于H+反向转运已牵连在运输周期中，我将定量表征H+结合LeuT的运输动力学的影响。此外，我将检查门控的Na+梯度，这是运输的生理驱动程序的调制。这些目标的完成将有助于阐明NSS的运输机制，可能通知下游的努力，筛选和/或设计新的治疗靶向NSS。此外，本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NSS）进行单分子成像。

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.