EXPRESSION CLONING OF A RESERPINE-SENSITIVE VESICULAR MONOAMINE TRANSPORTER
EXPRESSION CLONING OF A RESERPINE-SENSITIVE VESICULAR MONOAMINE TRANSPORTER
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DOI：
10.1073/pnas.89.22.10993
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发表时间：
1992-11-15
影响因子：
11.1
通讯作者：
HOFFMAN, BJ
中科院分区：
文献类型：
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作者：
ERICKSON, JD;EIDEN, LE;HOFFMAN, BJ
通过在哺乳动物细胞系(CV-1)中的表达克隆，获得了一个大鼠囊泡单胺转运蛋白的cDNA，命名为MAT。该序列预测了一个由515个氨基酸组成的蛋白质，有12个假定的跨膜结构域。表达该克隆的CV-1细胞积累[H-3]5-羟色胺的特性表明细胞内存在隔膜。在洋地黄素通透的细胞中，摄取依赖于ATP，表观K(M)为1.3um。质子转运型离子载体羰基氰化物对三氟甲氧基苯肼和液泡H+-ATPase抑制剂三(正丁基)锡可阻断摄取。抑制摄取的效力排序为：利血平、四苯肼、5-羟色胺、多巴胺、去甲肾上腺素、肾上腺素。[H-3]单胺摄取的直接比较表明，5-羟色胺是首选底物。用7-叠氮-8-[I-125]碘酮丝氨酸对表达MAT的CV-1细胞制备的膜进行光标记，发现主要的四苯并嗪敏感的光标记糖蛋白，其表观分子质量几乎等于-75 kDa。编码MAT的mRNA特异地存在于大鼠脑内蓝斑、黑质和中缝核的单胺细胞中，每种细胞都表达一种独特的质膜再摄取转运体。MAT基因克隆定义了一种囊泡单胺转运体，代表了一类不同的神经递质转运体分子。
A cDNA for a rat vesicular monoamine transporter, designated MAT, was isolated by expression cloning in a mammalian cell line (CV-1). The cDNA sequence predicts a protein of 515 amino acids with 12 putative membrane-spanning domains. The characteristics of [H-3]serotonin accumulation by CV-1 cells expressing the cDNA clone suggested sequestration by an intracellular compartment. In cells permeabilized with digitonin, uptake was ATP dependent with an apparent K(m) of 1.3 muM. Uptake was abolished by the proton-translocating ionophore carbonylcyanide p-trifluoromethoxyphenylhydrazone and with tri-(n-butyl)tin, an inhibitor of the vacuolar H+-ATPase. The rank order of potency to inhibit uptake was reserpine > tetrabenazine > serotonin > dopamine > norepinephrine > epinephrine. Direct comparison of [H-3]monoamine uptake indicated that serotonin was the preferred substrate. Photolabeling of membranes prepared from CV-1 cells expressing MAT with 7-azido-8-[I-125]iodoketanserin revealed a predominant tetrabenazine-sensitive photolabeled glycoprotein with an apparent molecular mass of almost-equal-to 75 kDa. The mRNA that encodes MAT was present specifically in monoamine-containing cells of the locus coeruleus, substantia nigra, and raphe nucleus of rat brain, each of which expresses a unique plasma membrane reuptake transporter. The MAT cDNA clone defines a vesicular monoamine transporter representing a distinct class of neurotransmitter transport molecules.