DOPAMINE TRANSPORTERS AND IONS, SUBSTRATES, BLOCKERS

描述(改编自申请人的摘要)： Na+、K+和Cl-在配基识别水平和 多巴胺转运蛋白(DAT)的底物转运。然而，准确的 关系是未知的，有许多关键的、未回答的问题 将在拟议的实验中解决的问题 遵循特定的目标。首先，将评估Na+是否刺激DA 通过在远端部位的变构效应与DAT结合，以及 作用是氯离子。我们之前的模拟研究表明，Na+位(阳离子位 2)可卡因类似物结合域的远端，在该结合域，Na+刺激可卡因 类比结合，以及不太确定的DA结合。第二，基材衍生 将研究DAT的抑制剂对Na+和Na+的差异敏感性 K+或对可卡因结合部位的优先亲和力与DA结合 网站。除了远端阳离子位点2之外，另一个位点(1)将是 研究了与DA领域重叠的内容，同样包括评估角色 对于氯离子，对于这些抑制剂。对于选定数量的衬底， DA与可卡因对Na+，K+的不同敏感性或优先作用 结合结构域，我们将探讨侧向性在抑制中的作用 DAT的外部和内部面孔，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 将根据文献中关于 Na+、Cl-和DA与DAT有序结合的序列。因为缺乏 在合适的Na+替代物中，[Na+]将以新的方案变化 没有替补的。经典动力学[~3H]DA摄取实验和旋转 在不同[Na+]和[Cl-]下的圆盘电极伏安测量将是 和预载的[~3H]DA的流出将作为以下函数进行检查 外排介质中的Na+和Cl-。第四，我们将在 DAT通过关注跨膜结构域参与Na+和Cl-的敏感性 (TMS)1、3和7。这些结构域中的残基与Na+和 与5-羟色胺和GABA转运体密切相关的CL-相互作用 与DAT相关。具体来说，我们希望评估Trp84和Arg85的作用 TM2中的Glu117，TM3中的Tyr156和Asn157，以及Asn353、Thr356、Ser357、 TM7中的Ser360、Gly361、Phe362、Phe365和leu368；此外，Cys90与 在TM1和2之间的细胞外环路中到TM1的关系将通过 定点突变。底物衍生化合物的联合探测 DAT蛋白中与A或残基相关的结构可能有助于确定 底物结构和DAT蛋白中的阳离子转化率区域 互动。总的来说，拟议的实验将改善我们的 理解离子与DAT相互作用对功能的影响 DAT在底物转运中的作用。

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.