Association of excitatory amino acid transporters, especially EAAT2, with cholesterol-rich lipid raft microdomains - Importance for excitatory amino acid transporter localization and function
Association of excitatory amino acid transporters, especially EAAT2, with cholesterol-rich lipid raft microdomains - Importance for excitatory amino acid transporter localization and function
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DOI：
10.1074/jbc.m403938200
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发表时间：
2004-08-13
影响因子：
4.8
通讯作者：
Lin, CLG
中科院分区：
文献类型：
--
作者：
Butchbach, MER;Tian, GL;Lin, CLG
在本研究中，我们研究了膜胆固醇在谷氨酸转运蛋白功能中的作用。甲基-β-环糊精消耗膜胆固醇导致原代皮质培养物中Na+依赖性谷氨酸吸收减少。胶质细胞谷氨酸转运蛋白 EAAT2 介导的摄取对此效应更敏感。细胞表面生物素化和免疫染色实验表明，胆固醇的损失显着改变了 EAAT2 向质膜的运输及其膜分布。在神经元谷氨酸转运蛋白 EAAT3 中也观察到了这些效应，但程度较小。此外，用甲基-β-环糊精处理小鼠脑质膜囊泡导致谷氨酸摄取显着减少，表明胆固醇消耗对谷氨酸转运蛋白的功能有直接影响。质膜胆固醇位于称为脂筏的离散微域内。对纯化的脂筏微结构域的分析表明，总 EAAT2 的大部分和总 EAAT1、EAAT3 和 EAAT4 的一小部分与脂筏相关。体内脂筏的人工聚集导致在细胞表面形成更大的 EAAT2 免疫反应簇。纯化的脂筏相关部分能够进行Na+依赖性谷氨酸吸收。我们的数据表明，谷氨酸转运蛋白，尤其是 EAAT2，与质膜富含胆固醇的脂筏微域相关，并且与这些富含胆固醇的微域的关联对于兴奋性氨基酸转运蛋白的定位和功能很重要。
In the present study, we investigated the role of membrane cholesterol in the function of glutamate transporters. Depletion of membrane cholesterol by methyl-beta-cyclodextrin resulted in reduced Na+-dependent glutamate uptake in primary cortical cultures. Glial glutamate transporter EAAT2-mediated uptake was more sensitive to this effect. Cell surface biotinylation and immunostaining experiments revealed that the loss of cholesterol significantly altered the trafficking of EAAT2 to the plasma membrane as well as their membrane distribution. These effects were also observed in neuronal glutamate transporter EAAT3 but to a lesser extent. Furthermore, the treatment of mouse brain plasma membrane vesicles with methyl-beta-cyclodextrin resulted in a significant reduction in glutamate uptake, suggesting that cholesterol depletion has a direct effect on the function of the glutamate transporters. Plasma membrane cholesterol is localized within discreet microdomains known as lipid rafts. Analyses of purified lipid raft microdomains revealed that a large portion of total EAAT2 and a minor portion of total EAAT1, EAAT3, and EAAT4 were associated with lipid rafts. Artificial aggregation of lipid rafts in vivo resulted in the formation of larger EAAT2-immunoreactive clusters on the cell surface. The purified lipid raft-associated fractions were capable of Na+-dependent glutamate uptake. Our data suggest that the glutamate transporters, especially EAAT2, are associated with cholesterol-rich lipid raft microdomains of the plasma membrane and that the association with these cholesterol-rich microdomains is important for excitatory amino acid transporter localization and function.