Ahn J, Mundigl O, Muth T R, Rudnick G, Caplan M J
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
J Biol Chem. 1996 Mar 22;271(12):6917-24. doi: 10.1074/jbc.271.12.6917.
At least three high affinity Na+- and Cl--dependent gamma-aminobutyric acid (GABA) transporters are known to exist in the rat and mouse brain. These transporters share 50-65% amino acid sequence identity with the kidney betaine transporter which also transports GABA but with lower affinity. The betaine transporter (BGT) is expressed on the basolateral surface of polarized Madin-Darby canine kidney (MDCK) cells. Recent evidence suggests that the signals and mechanisms involved in membrane protein sorting share many functional characteristics in polarized neurons and epithelial cells. It was previously shown that the rat GABA transporter GAT-1 is located in the presynaptic membrane of axons where it plays a role in terminating GABAergic neurotransmission. When expressed in MDCK cells by transfection, GAT-1 was sorted to the apical membrane. In this report, we have localized the other two GABA transporters, GAT-2 and GAT-3, in transfected MDCK cells by GABA uptake, immunofluorescence, and cell surface biotinylation. GAT-3, like GAT-1, localized to the apical membrane of MDCK cells while GAT-2, like BGT, localized to the basolateral membrane. We have also expressed BGT in low density cultures of hippocampal neurons by microinjection and immunolocalized it to the dendrites. The distribution of GAT-3 in these neurons after transfection was axonal as well as somatodendritic. These results indicate that highly homologous subtypes of GABA transporters are sorted differently when expressed in epithelial cells or neurons and suggest that these two cell types share the capacity to distinguish among these isoforms and target them to distinct destinations.
已知在大鼠和小鼠大脑中至少存在三种高亲和力的、依赖钠离子和氯离子的γ-氨基丁酸（GABA）转运体。这些转运体与肾脏中的甜菜碱转运体具有50 - 65%的氨基酸序列同一性，后者也能转运GABA，但亲和力较低。甜菜碱转运体（BGT）表达于极化的Madin-Darby犬肾（MDCK）细胞的基底外侧表面。最近的证据表明，参与膜蛋白分选的信号和机制在极化神经元和上皮细胞中具有许多功能特征。先前的研究表明，大鼠GABA转运体GAT-1位于轴突的突触前膜，在终止GABA能神经传递中发挥作用。通过转染在MDCK细胞中表达时，GAT-1被分选到顶端膜。在本报告中，我们通过GABA摄取、免疫荧光和细胞表面生物素化，在转染的MDCK细胞中定位了另外两种GABA转运体GAT-2和GAT-3。与GAT-1一样，GAT-3定位于MDCK细胞的顶端膜，而与BGT一样，GAT-2定位于基底外侧膜。我们还通过显微注射在海马神经元的低密度培养物中表达了BGT，并将其免疫定位到树突。转染后，GAT-3在这些神经元中的分布是轴突以及胞体树突状的。这些结果表明，GABA转运体的高度同源亚型在在上皮细胞或神经元中表达时的分选方式不同，这表明这两种细胞类型具有区分这些异构体并将它们靶向不同目的地的能力。
