Cloning of dopamine, norepinephrine and serotonin transporters from monkey brain: relevance to cocaine sensitivity
Cloning of dopamine, norepinephrine and serotonin transporters from monkey brain: relevance to cocaine sensitivity
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DOI：
10.1016/s0169-328x(00)00288-6
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发表时间：
2001-02-19
期刊：
影响因子：
--
通讯作者：
Madras, BK
中科院分区：
文献类型：
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作者：
Miller, GM;Yatin, SM;Madras, BK
我们利用RT-PCR技术克隆了猕猴、黄斑猕猴、猕猴和猕猴的单胺转运体（多巴胺转运体；DAT）和猕猴（去甲肾上腺素转运体；NET和血清素转运体；SERT）。猴的DAT、NET和SERT蛋白与人的同源性高达98%，在HEK-293细胞中表达时，显示出与猴脑或人单胺转运蛋白高度相关的药物亲和力和摄取动力学。与其他物种的报道相反，我们发现了双（苯基丙氨酸143的亮氨酸和谷氨酰胺509的精氨酸；变种I）和单（亮氨酸355的脯氨酸；变种LI）氨基酸变异。与与野生型DAT相同的[H-3] CFT （WIN 35,428）相比，变体I显示了多巴胺转运动力学和对各种DAT阻滞剂（包括可卡因）的结合亲和力（n = 7种药物；r(2)=0.991）。然而，我们检测到变体I （IC50: 488 +/- 102 nM， SEM, n = 3）和野生型DAT （IC50: 79 +/- 8.2 nM, n = 3, P < 0.05）对可卡因和[H-3]可卡因的亲和力差异为6倍。通过共聚焦显微镜检测，变体II定位于HEK-293细胞内，并且具有非常低的结合和多巴胺转运水平。还发现了一种新的NET外显子5剪接变体，其转运水平非常低，不与可卡因结合。通过NetPhos分析，我们在DAT、NET和SERT的细胞外环和细胞内环上检测到许多高度保守的推定磷酸化位点，这些位点可能对内化转运蛋白起作用。人和猴单胺转运蛋白的同源性和功能相似性进一步支持了灵长类动物在研究单胺转运蛋白在药物滥用机制、神经精神疾病和诊断和治疗药物开发中的作用的价值。(C) 2001 Elsevier Science B.V.版权所有
We used RT-PCR to clone monoamine transporters from Macaca mulatta, Macaca fasicularis, is and Saimiri sciureus (dopamine transporter; DAT) and Macaca mulatta (norepinephrine transporter; NET and serotonin transporter; SERT). Monkey DAT, NET and SERT proteins were >98% homologous to human and, when expressed in HEK-293 cells, displayed drug affinities and uptake kinetics that were highly correlated with monkey brain or human monoamine transporters. In contrast to reports of other species, we discovered double (leucine for phenylalanine 143 and arginine for glutamine 509; Variant I) and single (proline for leucine 355; Variant LI) amino acid variants of DAT. Variant I displayed dopamine transport kinetics and binding affinities for various DAT blockers (including cocaine) versus [H-3] CFT (WIN 35, 428) that were identical to wild-type DAT (n = 7 drugs; r(2)=0.991). However, we detected a six-fold difference in the affinity of cocaine versus [H-3] cocaine between Variant I (IC50: 488 +/- 102 nM, SEM, n = 3) and wild-type DAT (IC50: 79 +/- 8.2 nM, n = 3, P < 0.05). Variant II was localized intracellularly in HEK-293 cells, as detected by confocal microscopy, and had very low levels of binding and dopamine transport. Also discovered was a novel exon 5 splice variant of NET that displayed very low levels of transport and did not bind cocaine. With NetPhos analysis, we detected a number of highly conserved putative phosphorylation sites on extracellular as well as intracellular loops of the DAT, NET, and SERT, which may be functional for internalized transporters. The homology and functional similarity of human and monkey monoamine transporters further support the value of primates in investigating the role of monoamine transporters in substance abuse mechanisms, neuropsychiatric disorders and development of diagnostic and therapeutic agents. (C) 2001 Elsevier Science B.V. All rights reserved.