Bridging Project 4: Transport Cycle in Neurotransmitter Uptake Systems

DAT、NET和SERT是许多影响脑功能的药理学药物的既定靶点[16]。这些生物胺转运体通过将从突触裂隙释放的神经递质重新摄取回突触前神经元，再加上钠沿其电化学梯度移动来终止突触传递。干扰再摄取的药物会深刻地影响行为和情绪。为 例如，DAT是可卡因、安非他明和哌酸甲酯等精神兴奋剂的主要靶点[17]，而SERT的抑制剂是抗抑郁药(丙咪嗪、氟西芬)[18]。然而，我们对这些抑制剂发挥作用的分子机制的理解只停留在最初的五个阶段。 我们对Leut结构的分析[2]表明，有趣的是，结构和功能特征与我们目前对哺乳动物同源物DAT、SERT和Net的理解具有很强的一致性[1]。从建模的角度来看，这一点很重要，因为计算模拟结果强烈地受同源模型的质量的影响，而同源模型的质量又取决于保守的程度和 模板和目标之间的相似性。与真核生物相比，使用细菌膜蛋白和细菌表达系统的优势包括更容易放大和提高蛋白表达水平，以及更有限的翻译后修饰和更均匀的材料。因此，我们建议使用Leut作为拟议研究的既定模型系统[13]。 为了监测蛋白质在类天然条件下的构象变化，即在蛋白质脂质体中，没有晶格力的构象选择性，我们将使用已建立的8DSL-EPR技术(见[15])。 这项技术需要将天然残基定点突变为半胱氨酸，以加入巯基特定的氮氧化物自旋标记。对自旋标记蛋白质的EPR分析产生了描述在蛋白质序列中的选定位置掺入的氮氧化物探针的局部环境的光谱限制。这些结构约束是由诸如自旋标记溶剂可及性等可观察性产生的，它描述了 探头与其他顺磁性试剂的碰撞频率。此外，两个自旋标记物之间的偶极耦合已被证明是确定全局空间约束的有效光谱标尺，可以提供堆积相互作用和磁区运动的细节[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]。当基于从结构定义的或同源系统的动力学计算分析中产生的特定假设来选择标记点时，该方法成为在计算分析和模拟以及系统功能特性的实验数据的框架中确定结构-动力学环境中的关键功能特征的有力工具。拟议的研究针对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，即表征构成底物转位周期的构象状态(例如，见[11])。 由于没有多种构象状态的晶体结构(见上文)，需要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项胶水赠款提供了重大优势，因为它将利用核心(见下文第4.4节)和同源国的能力和资源 在本申请中描述的其他膜蛋白系统的研究。

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.