The Neurotransmitter: Sodium Symporter Permeation Pathway

描述（由申请人提供）：生物胺转运体在其特定神经递质底物的作用和回收中发挥重要作用。它们已被确定为许多影响脑功能的药理学药物的靶点。例如，可卡因的有益特性主要是由于其对多巴胺转运蛋白（DAT）的抑制；丙咪嗪的抗抑郁作用作用于血清素转运体（SERT）。这些转运蛋白属于神经递质钠同质转运蛋白（NSS）家族，该家族具有大量的原核同源物，包括色氨酸转运蛋白（TnaT）、酪氨酸转运蛋白（Tyt1）和亮氨酸转运蛋白（LeuT）。最近，一种高分辨率的Leu结构得到了求解，其衬底Leu被束缚在一个封闭的腔中。然而，对底物特异性的决定因素的理解对合理的药物设计很重要，它可能不仅存在于LeuT结构揭示的结合位点缝隙中，还存在于渗透途径中。本研究的长期目标是阐明NSS家族蛋白在易位周期中渗透途径的动态结构变化，并评估相互竞争的转运模型，如交替通路方案和底物跳跃模型。具体来说，使用原核生物nss蛋白、TnaT、Tyt1和LeuT作为模型系统，这将通过分子模型模拟和实验研究/验证之间的综合、比较迭代过程来实现。通过这一多学科协议，我们将探索沿渗透途径排列并在不同构象状态下动态重组的保守残基位置和潜在辅助空腔。将比较TnaT/Tyt1/LeuT以及其他原核和真核nss蛋白在渗透途径上的共同和不同特征，包括钠离子的作用。所获得的知识应该阐明生物胺转运体的转运周期，并将有助于我们对底物特异性的结构基础的理解。

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.