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）家族包括血清素、多巴胺和去甲肾上腺素的转运体，这些转运体是抗抑郁药、哌甲酯（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，针对GABA和甘氨酸的NSS分别是治疗癫痫、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的复杂相互作用调控。阐明NSS功能的分子机制是充分了解NSS调控的必要基础，从而设计改进的治疗干预手段。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是一种依赖于钠的氨基酸转运体，在晶体学上以不同的构象被捕获。这些数据极大地促进了我们对NSS传输机制的理解。然而，这些LeuT的静态“快照”无法揭示对理解NSS调控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细胞内表面的门控动力学的底物和抑制剂依赖调制。这些数据提供了关于转运机制的关键假设，我们现在的目标是通过首次成像LeuT细胞外表面的动力学来测试和探索。这些研究将使我们能够更深入地了解底物和抑制剂进入转运体的细胞外表面构象事件的作用，以及这些事件与调节底物释放的细胞内表面观察到的动力学之间的关系。我建议描述LeuT细胞内和细胞外动力学之间的关系，以及配体对其的调节。由于H+反端口与输运周期有关，我将定量表征H+与LeuT结合对输运动力学的影响。此外，我将研究由Na+梯度调制的门控，这是运输的生理驱动。这些目标的完成将有助于阐明NSS转运机制，潜在地为下游筛选和/或设计针对NSS的新疗法提供信息。此外，本提案中开发的单分子方法将有助于建立其他膜蛋白（包括人类NSS）在细胞膜上原位的单分子成像平台。

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.