Cell-specific sorting of biogenic amine transporters expressed in epithelial cells
Cell-specific sorting of biogenic amine transporters expressed in epithelial cells
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DOI：
10.1074/jbc.271.30.18100
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发表时间：
1996-07-26
影响因子：
4.8
通讯作者：
Rudnick, G
中科院分区：
文献类型：
--
作者：
Gu, HH;Ahn, J;Rudnick, G
我们利用稳定表达神经递质转运蛋白的极化上皮细胞来分析这些膜蛋白的分选行为。血清素 (5-HT)、多巴胺 (DA) 和去甲肾上腺素 (NE) 的转运蛋白预计原位存在于轴突膜的最远端，在那里它们终止其生物胺底物的作用。 Madin-Darby 犬肾 (MDCK) 和 LLC-PK1 细胞均用编码大鼠 5-HT 转运蛋白 (SERT)、人 NE 转运蛋白 (NET) 或大鼠或人 DA 转运蛋白 (DAT) 的 cDNA 稳定转染。这些细胞在渗透性过滤器支持物上生长，并且通过三种独立的技术对转运蛋白进行定位。共聚焦免疫荧光显微镜表明，LLC-PK1 细胞中表达的每种转运蛋白均被分选至基底外侧膜，与 Na+/K+-ATP 酶共定位。然而，在 MDCK 细胞中，DAT 主要位于顶端表面，而 SERT 和 NET 在基底外侧膜上发现，使用非渗透性生物素化试剂的细胞表面生物素化证实了免疫细胞化学结果，因此，MDCK 细胞中的 SERT 和 NET 在基底外侧培养基中比在顶端培养基中标记更有效，MDCK 细胞中的 DAT 在顶端侧标记比基底外侧更有效。转染的 MDCK 细胞与免疫细胞化学和生物素化结果一致，这些结果表明存在区分神经递质转运蛋白表面表达的细胞特异性机制，并且不可能做出任何简单的假设，即神经元和上皮细胞中的分选机制是相同的。
We have utilized polarized epithelial cells stably expressing neurotransmitter transporters to analyze the sorting behavior of these membrane proteins. The transporters for serotonin (5-HT), dopamine (DA), and norepinephrine (NE) are expected to be present in situ in the most distal extremities of axonal membranes, where they terminate the action of their biogenic amine substrates. Both Madin-Darby canine kidney (MDCK) and LLC-PK1 cells were stably transfected with cDNAs encoding either the rat 5-HT transporter (SERT), the human NE transporter (NET), or the rat or human DA transporter (DAT), These cells were grown on permeable filter supports, and the transporters were localized by three independent techniques, Confocal immunofluorescence microscopy indicated that each of the transporters expressed in LLC-PK1 cells was sorted to the basolateral membrane, co-localizing with the Na+/K+-ATPase. In MDCK cells, however, DAT was located primarily on the apical surface, while SERT and NET were found on the basolateral membranes, Cell surface biotinylation using an impermeant biotinylating reagent confirmed the immunocytochemistry results, Thus, SERT and NET in MDCK cells were labeled more efficiently from the basolateral medium than the apical medium, and DAT in MDCK cells was labeled more efficiently from the apical side than the basolateral side, Transport measurements in transfected MDCK cells agreed with the immunocytochemistry and biotinylation results, These results suggest the existence of cell-specific mechanisms that discriminate between neurotransmitter transporters for surface expression and render unlikely any simple hypothesis that sorting mechanisms in neurons and epithelia are identical.