Dopamine Transporter Structure-Function Studies

描述(申请人提供)：可卡因通过与多巴胺转运体(DAT)结合，阻断突触多巴胺的摄取，在大脑中启动其兴奋效应。到目前为止，还没有确定特定的DAT-配体接触，但人们普遍认为(由于先前的报告)，DAT跨膜(TM)1天冬氨酸残基(D79)与多巴胺和可卡因中的带电氮原子形成离子相互作用，控制着配体的识别。或者，D79残基可以形成配体芳香族结合口袋，即影响配体结合的离子渗透孔，或者可以简单地形成保留DAT基础结构的分子内接触。为了阐明DAT底物和抑制物结合部位的性质，关键是要确定DAT-配体相互作用的“离子对”模型是否可信。这项建议的目的是解决离子对模型的有效性，同时也评估D79残基对DAT结构和功能的替代潜在贡献。改变侧链大小、电荷或氢键潜力的D79 DAT突变体将通过一系列不同的DAT底物和抑制剂进行药理学表征。其他半胱氨酸取代的DAT突变体将测试半胱氨酸特异性烷基化试剂对DAT多肽中给定位置的可及性，以及这种烷基化事件是否影响DAT功能。通过测量引入的DAT TM 1(或附近)半胱氨酸残基(可变位置)在底物或抑制剂存在和不存在的情况下的可及性，将探索D79和DAT底物和抑制剂之间的空间关系。由于TM1天冬氨酸被认为是底物渗透孔的贡献者，因此也将使用取代半胱氨酸可及性方法来阐述TM1在D79附近的二级结构和细胞膜取向，对于该二级结构和细胞膜取向有多种模型。拟议的实验将显著提高对TM-1天冬氨酸残基在DAT功能中作用的理解，并推进对共享该残基的质膜去甲肾上腺素和5-羟色胺转运体的结构-功能研究。阐明这种残基在识别多巴胺和可卡因中的作用可能会推动合理的治疗设计，在不显著干扰多巴胺摄取的情况下阻止可卡因的作用。

DESCRIPTION (provided by applicant): Cocaine initiates its euphoric effects in the brain by binding to the dopamine transporter (DAT), blocking uptake of synaptic dopamine. No specific DAT-ligand contacts have been identified to date, but it is widely held (owing to a previous report) that the DAT transmembrane (TM) 1 aspartic acid residue (D79) forms an ionic interaction with charged nitrogen atoms in both dopamine and cocaine that governs recognition of the ligand. Alternatively, the D79 residue may contribute to a ligand aromatic binding pocket, an ion permeation pore that influences ligand binding, or may simply form intramolecular contacts that retain DAT infrastructure. Toward the goal of elaborating on the nature of DAT substrate and inhibtor binding sites, it is critical to determine whether the "ion pair" model for DAT-ligand interactions is credible. The objective of this proposal is to address the validity of the ion pair model, while also assessing alternative potential contributions of the D79 residue to DAT structure and function. D79 DAT mutants that modify side chain size, charge or hydrogen bonding potential will be pharmacologically characterized with a diverse collection of DAT substrates and inhibitors. Other, cysteine-substituted, DAT mutants will test the accessibility of a given position in the DAT polypeptide to cysteine-specific alkylating agents, and whether such alkylation events influence DAT function. By measuring accessibility of an introduced DAT TM 1 (or vicinity) cysteine residue (variably located) in the presence and absence of substrates or inhibitors, the spatial relationship between D79 and DAT substrates and inhibitors will be explored. Because the TM 1 aspartate has been implicated as a contributor to the substrate permeation pore, the substituted cysteine accessibility method will also be used to elaborate on the secondary structure and cell membrane orientation of TM 1 in the vicinity of D79, for which there are multiple models. The proposed experiments should significantly enhance understanding of the TM 1 aspartate residue role in DAT function, as well as advance structure-function studies on the plasma membrane norepinephrine and serotonin transporters which share this residue. Clarification of the role of this residue in recognition of dopamine and cocaine may forward rational design of therapeutics that block cocaine action without significantly interfering with dopamine uptake.