Brain glutamate transporter proteins form homomultimers
Brain glutamate transporter proteins form homomultimers
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DOI：
10.1074/jbc.271.44.27715
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发表时间：
1996-11-01
影响因子：
4.8
通讯作者：
Danbolt, NC
中科院分区：
文献类型：
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作者：
Haugeto, O;Ullensvang, K;Danbolt, NC
细胞外液中兴奋性氨基酸的去除对于突触传递和避免兴奋性毒性是必不可少的，这种去除是通过位于神经元和星形胶质细胞质膜上的谷氨酸转运体来完成的。摄取系统由几种不同的转运蛋白组成，这些转运蛋白受到仔细的调控，表明比简单的递质失活更精细的功能。在这里，我们通过化学交联，然后是电泳和免疫印迹，显示了三种大鼠脑谷氨酸转运蛋白(GLAST，GLT和EAAC)形成了同源多聚体。多聚体不仅存在于完整的脑膜中，而且在脂质体中溶解和重组后也存在，增加交联剂浓度增加了与三聚体相对应的条带的免疫反应性，但牺牲了二聚体和单体条带。然而，二聚体条带的免疫活性并没有消失，这表明存在二聚体和三聚体的混合。GLT和GLAST并不是相互复杂的，但包埋后的电子显微镜免疫细胞化学双标显示，它们并存于同一星形细胞细胞膜中，在体内寡聚体以非共价方式结合在一起。在体外，氧化诱导低聚物亚单位之间形成共价键(可能是-S-S-)，导致在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法上出现低聚物带。免疫沉淀实验表明，GLT是大脑中数量占主导地位的谷氨酸转运体。辐射失活分析给出了与低聚物结构相对应的功能复合体的分子靶标尺寸。我们假设谷氨酸转运体作为同源多聚体发挥作用。
Removal of excitatory amino acids from the extracellular fluid is essential for synaptic transmission and for avoiding excitotoxicity, The removal is accomplished by glutamate transporters located in the plasma membranes of both neurons and astroglia. The uptake system consists of several different transporter proteins that are carefully regulated, indicating more refined functions than simple transmitter inactivation, Here we show by chemical cross-linking, followed by electrophoresis and immunoblotting, that three rat brain glutamate transporter proteins (GLAST, GLT and EAAC) form homomultimers. The multimers exist not only in intact brain membranes but also after solubilization and after reconstitution in liposomes, Increasing the crosslinker concentration increased the immunoreactivity of the bands corresponding to trimers at the expense of the dimer and monomer bands. However, the immunoreactivities of the dimer bands did not disappear, indicating a mixture of dimers and trimers. GLT and GLAST do not complex with each other, but as demonstrated by double labeling post-embedding electron microscopic immunocytochemistry, they co-exist side by side in the same astrocytic cell membranes, The oligomers are held together noncovalently in vivo. In vitro, oxidation induces formation of covalent bonds (presumably -S-S-) between the subunits of the oligomers leading to the appearance of oligomer bands on SDS-polyacrylamide gel electrophoresis. Immunoprecipitation experiments suggest that GLT is the quantitatively dominant glutamate transporter in the brain. Radiation inactivation analysis gives a molecular target size of the functional complex corresponding to oligomeric structure. We postulate that the glutamate transporters operate as homomultimeric complexes.