Dersch C M, Akunne H C, Partilla J S, Char G U, de Costa B R, Rice K C, Carroll F I, Rothman R B
Clinical Psychopharmacology Section, NIDA Addiction Research Center, NIH, Baltimore, Maryland 21224.
Neurochem Res. 1994 Feb;19(2):201-8. doi: 10.1007/BF00966817.
The present study addressed the hypothesis that the DA transporter ligand, [3H]mazindol, labels multiple sites/states associated with the dopamine (DA) transporter in striatal membranes. Incubations with [3H]mazindol proceeded for 18-24 hr at 4 degrees C in 55.2 mM sodium phosphate buffer, pH 7.4, with a protease inhibitor cocktail. In order to obtain data suitable for quantitative curve fitting, it was necessary to repurify the [3H]mazindol by HPLC before a series of experiments. Under these conditions, we observed greater than 80% specific binding. The method of binding surface analysis was used to characterize the interaction of GBR12935, BTCP, mazindol, and CFT with binding site/sites labeled by [3H]mazindol. A one site model fit the data as well as the two site model: Bmax = 16911 fmol/mg protein, Kd of [3H]mazindol = 75 nM, Ki of GBR12935 = 8.1 nM, Ki of CFT = 50 nM and Ki of BTCP = 44 nM. The inhibitory mechanism (competitive or noncompetitive) of several drugs (GBR12935, CFT, BTCP, cocaine, cis-flupentixol, nomifensine, WIN35,065-2, bupropion, PCP, and benztropine) was determined. All drugs inhibited [3H]mazindol binding by a competitive mechanism. Although the ligand-selectivity of the [3H]mazindol binding site indicates that it is the uptake inhibitor recognition site of the classic DA transporter, the quantitative differences among the ligand-selectivities of different radioligands for the same site suggest that each radioligand labels different overlapping domains of the DA uptake inhibitor recognition site. It is likely that development of domain-selective drugs may further our understanding of the DA transporter.
多巴胺（DA）转运体配体[³H]吗茚酮标记了纹状体膜中与DA转运体相关的多个位点/状态。在含有蛋白酶抑制剂混合物的55.2 mM磷酸钠缓冲液（pH 7.4）中，于4℃用[³H]吗茚酮孵育18 - 24小时。为了获得适合定量曲线拟合的数据，在一系列实验之前，有必要通过高效液相色谱法（HPLC）对[³H]吗茚酮进行重新纯化。在这些条件下，我们观察到特异性结合率大于80%。采用结合表面分析方法来表征GBR12935、BTCP、吗茚酮和CFT与[³H]吗茚酮标记的结合位点之间的相互作用。单一位点模型与双位点模型对数据的拟合效果相当：最大结合容量（Bmax）= 16911 fmol/mg蛋白，[³H]吗茚酮的解离常数（Kd）= 75 nM，GBR12935的抑制常数（Ki）= 8.1 nM，CFT的Ki = 50 nM，BTCP的Ki = 44 nM。测定了几种药物（GBR12935、CFT、BTCP、可卡因、顺式氟哌噻吨、诺米芬辛、WIN35,065 - 2、安非他酮、苯环己哌啶和苯海索）的抑制机制（竞争性或非竞争性）。所有药物均通过竞争性机制抑制[³H]吗茚酮的结合。尽管[³H]吗茚酮结合位点的配体选择性表明它是经典DA转运体的摄取抑制剂识别位点，但同一位点不同放射性配体的配体选择性之间的定量差异表明，每种放射性配体标记了DA摄取抑制剂识别位点的不同重叠区域。开发区域选择性药物可能会进一步增进我们对DA转运体的理解。
