Bridging Project 4: Transport Cycle in Neurotransmitter Uptake Systems

DAT、NET和SERT是许多影响脑功能的药物的公认靶点[16]。这些生物胺转运蛋白通过将释放的神经递质从突触间隙再摄取回到突触前神经元，结合Na+沿其电化学梯度的运动来终止突触传递。干扰重摄取的药物会深刻地影响行为和情绪。为 例如，DAT是精神兴奋剂可卡因、安非他明和哌醋甲酯的主要靶点[17]，而SERT的抑制剂是抗抑郁药（丙咪嗪、氟西汀）[18]。然而，我们对这些抑制剂发挥作用的分子机制的理解还处于一个新的阶段。 我们对LeuT结构的分析[2]显示了结构和功能特征与我们目前对哺乳动物同系物DAT，SERT和NET [1]的理解之间的强烈一致性。从建模的角度来看，这是重要的，因为计算模拟结果受到同源性模型的质量的强烈影响，而同源性模型的质量又取决于保守性的程度， 模板和目标之间的相似性。与细菌膜蛋白和细菌表达系统一起工作的优点包括更容易放大和增加蛋白质表达水平，以及更有限的翻译后修饰，因此与它们的真核对应物相比更均匀的材料。因此，我们建议使用LeuT作为拟定研究的既定模型系统[13]。 为了在天然样条件下监测蛋白质构象变化，即在蛋白脂质体中，没有晶格力的构象选择性，我们将使用已建立的8DSL-EPR技术（参见[15]）。 该技术需要将天然残基定点突变为半胱氨酸以掺入巯基特异性氮氧自由基自旋标记。自旋标记的蛋白质的EPR分析产生描述在蛋白质序列中的选择位点处并入的氮氧探针的局部环境的光谱约束。这些结构约束是由可观测量产生的，例如自旋标记溶剂可及性，它描述了 探针与其他顺磁试剂的碰撞频率。此外，两个自旋标记之间的偶极耦合已被证明是用于确定全局空间约束的有效光谱标尺，其可以提供堆积相互作用和域运动的细节[19]。这些光谱特征模式的变化已被证明与蛋白质结构的构象变化相关 [20]第20段。当标签网站选择的基础上产生的特定假设从computafional分析的动态结构定义或同源系统，该方法成为一个深刻的工具，用于确定关键功能特性的结构-动态的背景下，这是可解释的，反过来，在计算分析和模拟的框架和实验数据的系统的功能特性。所提出的研究旨在解决NSS或任何转运蛋白家族的结构-功能研究中的一个主要挑战，即表征构成底物易位循环的构象状态（例如，见[11]）。 缺乏多个构象状态的晶体结构（见上文）要求通过我们在这里提出的计算和实验研究相结合的方法来阐明运输的动态性质。在这方面，将拟议的研究纳入这一粘合补助金具有重大优势，因为它将利用核心研究（见下文第4.4节）和同源研究的能力和资源。 如本申请通篇所述的其它膜蛋白系统的研究。

DAT, NET, and SERT are well established targets for many pharmacological agents that affect brain function [16]. These biogenic amine transporters terminate synapfic transmission by reuptake of the released neurotransmitters from the synaptic cleft back to the presynaptic neuron, coupled to the movement of Na+ down its electrochemical gradient. Drugs that interfere with reuptake profoundly influence behavior and mood. For example, DAT is the primary target for the psychosfimulants cocaine, amphetamine, and methylphenidate [17] whereas inhibitors of SERT are antidepressants (imipramine, fluoxefine) [18]. However, our understanding of the molecular mechanisms whereby these inhibitors exert their effects is sfill at a primifive stage. Our analysis of the LeuT structure [2] has shown intriguingly strong consistency between the structural and funcfional characteristics and our current understanding of the mammalian homologues DAT, SERT, and NET [1]. This is important from a modeling perspective, because computational simulation results are strongly influenced by the quality of the homology models, which in turn depends on the degree of conservafion and similarity between the template and target. Advantages to working with bacterial membrane proteins and bacterial expression systems include easier scale up and increased levels of protein expression, and more limited posttranslational modificafion and thus more homogeneous material as compared to their eukaryotic counterparts. Thus, we propose to use LeuT as an established model system [13] for the proposed studies. To monitor protein conformational changes under native-like conditions, i.e. in proteoliposomes, without the conformational selectivity of crystal lattice forces, we will use the established 8DSL-EPR technique (see [15]). This technique requires site-directed mutafion of native residues to cysteine for the incorporation of a sulfhydryl-specific nitroxide spin label. EPR analysis of the spin labeled proteins yields spectroscopic constraints describing the local environment of a nitroxide probe incorporated at select sites in a protein sequence. These structural constraints are generated from observables such as spin label solvent accessibility, which describes the collisional frequency of the probe with other paramagnetic reagents. Furthermore, dipolar coupling between two spin labels has been shown to be an effective spectroscopic ruler for the determination of global spatial constraints that can provide details of packing interactions and domain movements[19]. Changes in the pattern of these spectroscopic signatures have been shown to correlate with conformafional changes in protein structure [20]. When the labeling sites are selected based on specific hypotheses generated from computafional analyses of dynamics in structurally defined or cognate systems, the approach becomes an incisive tool for determining key functional characteristics in a structure-dynamic context that is interpretable, in turn, in the frame of the computational analysis and simulation and the experimental data for functional properties of the system. The proposed studies aim at a major challenge in structure-function studies of NSS or any transporter family, namely the characterization of the conformational states that constitute the substrate translocation cycle (e.g., see [11]). The absence of crystal structures for multiple conformational states (see above) calls for elucidation of the dynamic nature of transport through the type of combined approach of computational and experimental studies we propose here. In this respect, the integration of the proposed study in this glue grant offers major advantages as it will take advantage of the capabilities and resources in the Cores (see section 4.4, below) and the cognate studies on other membrane protein systems as described throughout this application.