Eshleman A J, Neve R L, Janowsky A, Neve K A
Research Service, Veterans Affairs Medical Center, Portland, Oregon, USA.
J Pharmacol Exp Ther. 1995 Jul;274(1):276-83.
A 3.5-kilobase cDNA encoding the dopamine transporter was isolated from a human substantia nigra cDNA library. Sequence analysis of the coding region of the transporter identified two nucleotide differences between the cDNA and published human dopamine transporter sequences. One of the substitutions changed an amino acid conserved among previously cloned dopamine (DA) and norepinephrine transporters, Arg-344, to a methionine. C6 glioma cells or COS-7 cells transfected with the cDNA (C6-hDAT and Cos7-hDAT cells) accumulated [3H]DA with high affinity (Km = 1.2 and 1.5 microM, respectively), and DA uptake inhibitors had similar potencies in both cell lines. [3H]2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane ([3H]CFT) bound to membranes prepared from both cell lines with high affinity (Kd = 2-6 nM), although some experiments with C6-hDAT cell membranes indicated the presence of a second class of binding sites with lower affinity for the radioligand. Using the high-affinity Kd value for [3H]CFT binding determined in Cos7-hDAT cells to calculate Ki values, drug affinity for inhibition was highly correlated (r = .92) with affinity for inhibition of [3H]DA uptake, although transporter substrates were significantly more potent inhibitors of uptake than of [3H]CFT binding. The binding of [3H]1-[2-diphenylmethoxy]ethyl-4-(3-phenylpropyl)-piperazine ([3H]GBR-12935) to C6-hDAT cells could not be characterized due to high binding to untransfected C6 cells, but on Cos7-hDAT cells the radioligand labeled a single population of binding sites (Kd = 1 nM). Inhibition of [3H]GBR-12935 binding by drugs correlated highly with inhibition of either [3H]CFT binding (r = .98) or of [3H]DA uptake (r = .95).(ABSTRACT TRUNCATED AT 250 WORDS)
从人黑质cDNA文库中分离出一个编码多巴胺转运体的3.5千碱基cDNA。对转运体编码区的序列分析确定了该cDNA与已发表的人多巴胺转运体序列之间存在两个核苷酸差异。其中一个替换将先前克隆的多巴胺（DA）和去甲肾上腺素转运体中保守的氨基酸精氨酸-344变为甲硫氨酸。用该cDNA转染的C6胶质瘤细胞或COS-7细胞（C6-hDAT和Cos7-hDAT细胞）以高亲和力积累[3H]DA（Km分别为1.2和1.5微摩尔），并且DA摄取抑制剂在两种细胞系中的效力相似。[3H]2β-甲氧羰基-3β-（4-氟苯基）托烷（[3H]CFT）以高亲和力（Kd = 2 - 6纳摩尔）与两种细胞系制备的膜结合，尽管对C6-hDAT细胞膜进行的一些实验表明存在对放射性配体亲和力较低的第二类结合位点。使用在Cos7-hDAT细胞中测定的[3H]CFT结合的高亲和力Kd值来计算Ki值，药物对抑制的亲和力与对[3H]DA摄取抑制的亲和力高度相关（r = 0.92），尽管转运体底物对摄取的抑制作用比对[3H]CFT结合的抑制作用明显更强。由于[3H]1-[2-二苯基甲氧基]乙基-4-（3-苯基丙基）-哌嗪（[3H]GBR-12935）与未转染的C6细胞结合力高，因此无法对其与C6-hDAT细胞的结合进行表征，但在Cos7-hDAT细胞上，该放射性配体标记了单一的结合位点群体（Kd = 1纳摩尔）。药物对[3H]GBR-12935结合的抑制与对[3H]CFT结合（r = 0.98）或[3H]DA摄取（r = 0.95）的抑制高度相关。（摘要截断于250字）
