Erickson J D, Eiden L E, Hoffman B J
Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, MD 20892.
Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10993-7. doi: 10.1073/pnas.89.22.10993.
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 [3H]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 Km of 1.3 microM. 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 [3H]monoamine uptake indicated that serotonin was the preferred substrate. Photolabeling of membranes prepared from CV-1 cells expressing MAT with 7-azido-8-[125I]iodoketanserin revealed a predominant tetrabenazine-sensitive photolabeled glycoprotein with an apparent molecular mass of approximately 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.
通过在哺乳动物细胞系（CV-1）中进行表达克隆，分离出了一种大鼠囊泡单胺转运体的互补DNA（cDNA），命名为MAT。该cDNA序列预测编码一个含有515个氨基酸的蛋白质，具有12个假定的跨膜结构域。表达该cDNA克隆的CV-1细胞对[³H]血清素的摄取特征表明其被细胞内区室隔离。在用洋地黄皂苷通透处理的细胞中，摄取依赖于ATP，表观Km为1.3微摩尔。质子转运离子载体羰基氰化物对三氟甲氧基苯腙和液泡H⁺-ATP酶抑制剂三（正丁基）锡可消除摄取。抑制摄取的效力顺序为利血平＞丁苯那嗪＞血清素＞多巴胺＞去甲肾上腺素＞肾上腺素。对[³H]单胺摄取的直接比较表明血清素是首选底物。用7-叠氮基-8-[¹²⁵I]碘酮舍林对表达MAT的CV-1细胞制备的膜进行光标记，显示出一种主要的对丁苯那嗪敏感的光标记糖蛋白，表观分子量约为75 kDa。编码MAT的mRNA特异性存在于大鼠脑蓝斑、黑质和中缝核中含单胺的细胞中，每个细胞都表达一种独特的质膜再摄取转运体。MAT cDNA克隆定义了一种囊泡单胺转运体，代表了一类独特的神经递质转运分子。
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