The dopamine transporter and ions, substrates, blockers

描述(申请人提供)：在本项目的前一阶段(由DA 13261-01至05支持)，我们已经研究了Na+和Cl-对多巴胺(DA)与人DA转运蛋白(HDAT)结合的影响以及膜电位和跨膜离子梯度的作用；底物样化合物在DAT上对可卡因与DA结合区域有一定的偏好；以及DAT残基参与离子敏感性，特别是保守的Trp和酸性残基。现在，我们希望进一步表征控制类可卡因、苯并托品/GBR 12909类和底物类化合物与DAT相互作用的结构特征，并进一步研究转运蛋白的氯离子调节，如下所示。(1)评价苯并托品/GBR 12909分子的结构特征(S)，以区分其与可卡因的结合。我们将利用我们以前开发的W84L和D313N突变体，结合可卡因、苯并托品和GBR 12909的结构类似物，一方面研究CFT/可卡因与苯妥拉平/GBR 12909之间的差异，另一方面，在与DAT相互作用时，我们将研究CFT/可卡因和苯妥拉平/GBR 12909之间的差异。还将评估这些抑制剂的结构-活性关系(SAR)，以确定它们是否能够改变DAT半胱氨酸残基的化学修饰，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的突变来定位与某些DAT残基相互作用的可卡因(DA)基团。我们将研究可卡因中的羧基(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，或者氮与芳烃之间的阳离子-pi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；也将评估对DAT转运的影响。我们将集中于DA类化合物，添加邻苯二酚的氨基萘，以及二聚化的类DA或类萘结构的二价配体，它们可能与DAT二聚体结合。(4)研究氯离子在DAT功能中的作用，确定控制转运蛋白氯离子调控的保守的Arg和Lys残基。我们将通过我们以前开发的偏爱向外或向内状态的突变体以及通过不同浓度的Na+或Zn2+促进DAT构象变化来研究Cl-的作用。还将研究底物结合的氯离子依赖性。

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.