Rothman R B, Silverthorn M L, Baumann M H, Goodman C B, Cadet J L, Matecka D, Rice K C, Carroll F I, Wang J B, Uhl G R
Clinical Pharmacology Section, National Institute on Drug Abuse, National Institutes of Health, Baltimore, Maryland, USA.
J Pharmacol Exp Ther. 1995 Jul;274(1):385-95.
Previous studies showed that the cocaine analog [125I]RTI-55 labels dopamine and serotonergic (5-HT) biogenic amine transporters (BATs) with high affinity. Here we characterized [125I]RTI-55 binding to membranes prepared from whole rat brain minus the caudate nuclei. Paroxetine (50 nM) was used to block [125I]RTI-55 binding to 5-HT transporter sites. Initial experiments identified drugs that displaced [125I]RTI-55 binding with moderately low slope factors. Binding surface analysis of the interaction of 3 beta-(4-chlorophenyl)tropan-2 beta-carboxylic acid phenyl ester hydrochloride (RTI-113) and 3 beta-(4-iodophenyl)tropan-2 beta-carboxylic acid phenyl ester hydrochloride (RTI-122) with [125I]RTI-55 binding sites readily resolved two binding sites for [125I]RTI-55 with Kd values of 0.44 nM and 17 nM and Bmax values of 31 and 245 fmol/mg protein. Potent 5-HT and noradrenergic uptake inhibitors had low affinity for both sites. Whereas cocaine, CFT and WIN35,065-2 were 6.0-, 25- and 14-fold selective for the first site, benztropine, PCP and the novel pyrrole, (+-)-(2RS,3aSR,8bRS)-1,2,3,3a,4,8b-hexahydro- 2-benzyl-1-methylindeno-[1,2-b]pyrrole resorcylate [(+-)-HBMP, formerly called (+-)-RTI-4793-14], were moderately selective for the second site. A single binding site with the characteristics of site 1 was resolved using COS cells transiently expressing the cloned rat dopamine transporter. Lesion studies with 6-hydroxydopamine and 5,7-dihydroxytryptamine were conducted to test the hypothesis that site 1 and site 2 are physically distinct. The data showed that these neurotoxins differentially decreased [125I]RTI-55 binding to sites 1 and 2. The differential distribution of sites 1 and 2 in rat brain provides further support for this hypothesis. Viewed collectively, these data show that [125I]RTI-55 labels a novel binding site in rat brain membranes, termed DATsite2, which is not associated with the classic dopamine, serotonin or norepinephrine transporters.
先前的研究表明，可卡因类似物[125I]RTI-55能以高亲和力标记多巴胺和血清素能（5-HT）生物胺转运体（BATs）。在此，我们对[125I]RTI-55与从去除尾状核的大鼠全脑制备的膜的结合进行了表征。帕罗西汀（50 nM）用于阻断[125I]RTI-55与5-HT转运体位点的结合。初步实验确定了以适度低斜率因子取代[125I]RTI-55结合的药物。对3β-(4-氯苯基)托烷-2β-羧酸苯酯盐酸盐（RTI-113）和3β-(4-碘苯基)托烷-2β-羧酸苯酯盐酸盐（RTI-122）与[125I]RTI-55结合位点相互作用的结合表面分析很容易解析出[125I]RTI-55的两个结合位点，其Kd值分别为0.44 nM和17 nM，Bmax值分别为31和245 fmol/mg蛋白质。强效的5-HT和去甲肾上腺素摄取抑制剂对这两个位点的亲和力都很低。可卡因、CFT和WIN35,065-2对第一个位点的选择性分别为6.0倍、25倍和14倍，而苯海索、PCP和新型吡咯（(+-)-(2RS,3aSR,8bRS)-1,2,3,3a,4,8b-六氢-2-苄基-1-甲基茚并-[1,2-b]吡咯间苯二酚盐[(+-)-HBMP，原称为(+-)-RTI-4793-14]）对第二个位点有适度的选择性。使用瞬时表达克隆大鼠多巴胺转运体的COS细胞解析出了具有位点1特征的单个结合位点。用6-羟基多巴胺和5,7-二羟基色胺进行损伤研究，以检验位点1和位点2在物理上是不同的这一假设。数据表明，这些神经毒素对位点1和位点2的[125I]RTI-55结合有不同程度的降低。位点1和位点2在大鼠脑中的差异分布为这一假设提供了进一步支持。综合来看，这些数据表明[125I]RTI-55在大鼠脑膜中标记了一个新的结合位点，称为DATsite2，它与经典的多巴胺、血清素或去甲肾上腺素转运体无关。
