The Neurotransmitter: Sodium Symporter Permeation Pathway

描述(申请人提供)：生物胺转运体在其特定神经递质底物的作用和循环中发挥重要作用。它们已经被确定为许多影响大脑功能的药理学药物的靶标。例如，可卡因的奖赏特性主要是由于它抑制多巴胺转运体(DAT)；丙咪嗪的抗抑郁作用是通过抑制5-羟色胺转运体(SERT)来实现的。这些转运体属于神经递质钠转运体(NSS)家族，具有大量的原核同源基因，包括色氨酸转运体(TNAT)、酪氨酸转运体(Tyt1)和亮氨酸转运体(Leut)。最近，一种高分辨率的Leut结构被解决，衬底Leu束缚在一个封闭的腔中。然而，底物专一性的决定因素，对于合理的药物设计非常重要，可能不仅存在于Leut结构揭示的结合部位缝隙内，而且还存在于渗透途径上。本建议的长期目标是阐明NSS家族蛋白在易位周期中渗透途径的动态结构变化，并评价竞争性运输模型，如交替访问方案和底物跳跃模型。具体地说，使用原核生物NSS-蛋白质、TNAT、Tyt1和Leut作为模型系统，这将通过分子建模模拟和实验调查/验证之间的完整、比较的迭代过程来实现。通过这一多学科的方案，我们将探索保守的残基位置和潜在的辅助空洞，它们排列在渗透途径上，并在不同的构象状态下动态重组。我们将比较TNAT/Tyt1/Leut以及与其他原核和真核NSS-蛋白的共同和不同的渗透途径，包括钠离子的作用。所获得的知识应该有助于阐明生物胺转运体的转位循环，并有助于我们理解底物特异性的结构基础。

DESCRIPTION (provided by applicant): Biogenic amine transporters play important roles in the action and recycling of their specific neuretransmitter substrates. They have been well established as the targets for many pharmacological agents that affect brain function. For examples, the rewarding properties of cocaine are due mainly to its inhibition of dopamine transporter (DAT); the antidepressant effect of imipramine is exerted on serotonin transporter (SERT). These transporters belong to the NeurotransmitterSodium Symporter (NSS) family, which has a large number of prokaryotic homologs, including a tryptophan transporter (TnaT), a tyrosine transporter (Tyt1), and a leucine transporter (LeuT). Recently, a LeuT structure has been solved in high resolution, with the substrate Leu bound in an enclosed cavity. However, the determinants of substrate specificity, an understanding of which is important for rational drug design, may lie not only within the binding site crevice revealed by the LeuT structure, but also along the permeation pathway. The long term goal of the present proposal is to elucidate dynamic structural changes of the permeation pathway of NSS family proteins during the translocation cycle and to evaluate competing transport models, e. g., the alternating-access scheme and the substrate hopping model. Specifically, using prokaryotic NSS-proteins, TnaT, Tyt1, and LeuT as model systems, this will be achieved by an integrated, comparative process of iteration between molecular modeling simulations and experimental investigations/validations. With this multidisciplinary protocol we will explore the conserved residue positions and potential auxiliary cavities that line the permeation pathway and reorganize dynamically in different conformational states. The common, and the different features of the permeation pathways, including the roles of sodium ions, will be compared among TnaT/Tyt1/LeuT, and with other prokaryotic and eukaryotic NSS-proteins. The knowledge acquired should shed light on the translocation cycles of biogenic amine transporters and will contribute to our understanding of the structural bases of substrate specificities.