DOPAMINE TRANSPORTERS AND IONS, SUBSTRATES, BLOCKERS

描述（改编自申请人摘要）： Na+、K+和Cl-在配体识别水平上起着关键作用， 多巴胺转运蛋白（DAT）的底物转运。但具体 关系是未知的，有一些关键的，没有答案的， 将在《公约》下拟议的实验中解决的问题 遵循具体目标。首先，将评估Na+是否刺激DA 通过在远端位点的变构效应与DAT结合， 作用是Cl-。我们以前的建模研究表明，Na+位点（阳离子位点 2)在可卡因类似物结合结构域的远端，Na+刺激可卡因 类似物结合和DA结合。二、基质衍生 DAT的抑制剂将研究对Na+和 K+或对可卡因结合位点与DA结合具有优先亲和力 网站.除了远端阳离子位点2之外，另一个位点（1）将被 研究了与DA领域重叠的问题，再次包括评估 对于这些抑制剂的Cl-。对于选定数量的基板， 对DA和可卡因的Na+、K+敏感性差异或优先作用 结合域，我们将探讨在抑制的作用， DAT的外部和内部面，并评估其可卡因拮抗剂 活动第三，Na+、Cl-和DA在转运循环中的结合顺序 将研究鉴于文献中的分歧， 在Na+、Cl-和DA与DAT的有序结合中， Na+的合适替代物，[Na+]将在新方案中变化 没有替代品。经典动力学[3 H]DA摄取实验和旋转 将在不同的[Na+]和[Cl-]下进行圆盘电极伏安法测量， 进行，并将检查预载[3 H]DA的外排，作为 Na+和Cl-在流出介质中。第四，我们将寻找残留物， DAT通过跨膜结构域参与Na+和Cl-敏感性 (TMs)1、3和7。这些结构域中的残基与Na+和 Cl-与5-羟色胺和GABA的转运蛋白相互作用， 具体而言，我们希望评估Trp 84和Arg 85的作用 在TM 1中，TM 2中Glu 117，TM 3中Tyr 156和Asn 157，以及Asn 353，thr 356，Ser 357， TM 7中的Ser 360、Gly 361、Phe 362、Phe 365和leu 368;此外， 将通过以下方法研究TM 1和2之间的细胞外环中的TM 1 定点诱变。底物衍生的化合物的组合探测 DAT蛋白中与A或残基相关的结构可能有助于确定 在底物结构和DAT蛋白中， 互动总的来说，拟议的实验将改善我们的 理解离子与DAT的相互作用对功能的影响， 转移底物中的DAT。

DESCRIPTION(Adapted from applicant's abstract): Na+, K+, and Cl- play a key role, both at the level of ligand recognition and substrate translocation by the dopamine transporter (DAT). However, the exact relationships are not known, and there are a number of crucial, unanswered questions that will be addressed in the proposed experiments under the following Specific Aims. First, it will be assessed whether Na+ stimulates DA binding to the DAT through an allosteric effect at a distal site, and what the role is of Cl-. Our previous modeling studies indicated a Na+ site (cation site 2) distal to the cocaine analog binding domain, at which Na+ stimulates cocaine analog binding, and, less certainly, DA binding. Second, substrate-derived inhibitors of the DAT will be studied with differential sensitivity to Na+ and K+ or with preferential affinity for cocaine binding sites vs. DA binding sites. In additions to the distal cation site 2, another site (1) will be studied that overlaps with the DA domain, again including assessment of a role for Cl- for these inhibitors. For a selected number of substrates with differential Na+, K+ sensitivity or with preferential action at DA vs. cocaine binding domains, we will explore the role of sidedness of inhibition at the external andinternal face of the DAT, and assess their cocaine antagonist activity. Third, the order of binding of Na+, Cl- and DA in the transport cycle will be studied in view of the disagreement in the literature as to the sequence in the ordered binding of Na+, Cl- and DA to DAT. Because of the lack of a suitable substitute for Na+, [Na+] will be varied in a novel protocol without a substitute. Classic kinetic [3H]DA uptake experiments and rotating disk electrode voltammetry measurements at varying [Na+] and [Cl-] will be performed and the efflux of preloaded [3H]DA will be examined as a function of Na+ and Cl- in the efflux medium. Fourth, we will search for residues in the DAT involved in Na+ and Cl- sensitivity, by focusing on transmembrane domains (TMs) 1, 3, and 7. Residues in these domains have been implicated in Na+ and Cl- interactions with the transporters for serotonin and GABA which are closely related to the DAT. Specifically, we wish to assess the role of Trp84 and Arg85 in tm1, Glu117 in TM2, Tyr156 and Asn157 in TM3, and Asn353, thr356, Ser357, Ser360, Gly361, Phe362, Phe365, and leu368 in TM7; in addition, Cys90 adjacent to TM1 in the extracellular loop between TM1 and 2 will be studied by site-directed mutagenesis. Combined probing of substrate-derived compounds of structures related to A or residues in the DAT protein may help to define regions in both the substrate structure and the DAT protein that rate to cation interaction. Collectively, the proposed experiments will improve our understanding of ion interactions with the DAT impacting on the functioncof the DAT in translocating substrate.