The dopamine transporter and ions, substrates, blockers

项目前期（由申请人提供），我们研究了Na+和Cl-对多巴胺（DA）与人DA转运体（hDAT）结合的影响，以及膜电位和跨膜离子梯度的作用；在DAT上对可卡因和DA结合域具有一定偏好的底物样化合物；和参与离子敏感性的DAT残基，特别是保守的色氨酸和酸性残基。现在，我们希望进一步表征可卡因样、苯托品/GBR 12909样和底物样化合物与DAT相互作用的结构特征，并进一步研究转运体的Cl-调节，如下所示。(1)评价苯托品/GBR 12909分子与可卡因结合的结构特征。我们将利用我们之前开发的W84L和D313N突变体，结合可卡因、苯托品和GBR 12909的结构类似物，研究CFT/可卡因与苯托品/GBR 12909在与DAT相互作用方面的差异。这些抑制剂的结构-活性关系（SAR）也将被评估，因为它们能够改变DAT Cys残基的化学修饰，并通过运输效应增加DAT的表面存在。(2)通过结合可卡因（DA）的结构变化和DAT的突变来确定可卡因（DA）与某些DAT残基相互作用的群体。我们将研究可卡因中羧基（或DA中的羟基）与DAT中酪氨酸或丝氨酸的羟基之间的氢键作用，或氮与芳烃之间的阳离子- π相互作用。(3)研究底物衍生化合物作为底物、抑制剂或潜在的可卡因拮抗剂与DAT相互作用时的SAR；还将评估对数据贩运的影响。我们将重点关注da样化合物、添加儿茶酚的氨基萘，以及可能与DAT二聚体结合的da样或萘样结构的二价配体。(4)研究Cl-在DAT功能中的作用，确定控制转运体Cl-调制的保守的Arg和Lys残基。我们将研究Cl-与我们之前开发的突变体的作用，这些突变体倾向于向外或内向的状态，以及不同浓度的Na+或Zn2+促进DAT构象变化。底物结合的cl依赖性也将被研究。

DESCRIPTION (provided by applicant): In the previous stage of this project (supported by DA 13261-01 through 05), we have studied the impact of Na+ and Cl- on dopamine (DA) binding to the human DA transporter (hDAT) and the role of membrane potential along with transmembrane ion gradients; substrate-like compounds with some preference for the cocaine vs. DA binding domain on the DAT; and DAT residues involved in ion sensitivity, in particular conserved Trp and acidic residues. Now we wish to further characterize the structural features governing the interaction of cocaine-like, benztropine/GBR 12909-like, and substrate-like compounds with DAT, and further study Cl- modulation of the transporter, as follows. (1) To assess the structural feature(s) of the benztropine/GBR 12909 molecule distinguishing its binding from that of cocaine. We will study the difference between CFT/cocaine, on the one hand, and benztropine/GBR 12909, on the other hand, in interacting with DAT, using our previously developed W84L and D313N mutants, combined with structural analogs of cocaine, benztropine, and GBR 12909. The structure-activity relationships (SAR) for these inhibitors will also be assessed for their ability to alter chemical modification of DAT Cys residues and to increase the surface presence of DAT by trafficking effects. (2) To pinpoint groups of cocaine (DA) interacting with certain DAT residues by combining structural changes in cocaine (DA) with mutations in DAT. We will study the role of hydrogen bonding between carboxyl groups in cocaine (or hydroxyls in DA) and hydroxyls of tyrosine or serine in DAT, or cation-pi interaction between nitrogen and aromatics. (3) To study the SAR of substrate-derived compounds in interacting with DAT as a substrate, inhibitor, or potential cocaine antagonist; effects on DAT trafficking will also be assessed. We will focus on DA-like compounds, aminonaphthalenes with added catechol, and bivalent ligands of dimerized DA-like or naphthalene-like structures, potentially binding to DAT dimers. (4) To study the role of Cl- in DAT function and identify conserved Arg and Lys residues controlling Cl- modulation of the transporter. We will study the role of Cl- with our previously developed mutants that prefer either the outward- or inward-facing state as well as by varying concentrations of Na+ or Zn2+ that promote DAT conformational changes. The Cl-dependence of substrate binding will also be studied.