High-affinity glutamate transporter GLAST/EAAT1 regulates cell surface expression of glutamine/neutral amino acid transporter ASCT2 in human fetal astrocytes
High-affinity glutamate transporter GLAST/EAAT1 regulates cell surface expression of glutamine/neutral amino acid transporter ASCT2 in human fetal astrocytes
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DOI：
10.1016/j.neuint.2005.12.033
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发表时间：
2006-05-01
影响因子：
4.2
通讯作者：
Gegelashvili, G
中科院分区：
文献类型：
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作者：
Gegelashvili, M;Rodriguez-Kern, A;Gegelashvili, G
中性氨基酸转运蛋白ASCT 2与高亲和力谷氨酸转运蛋白一起属于钠离子依赖性溶质转运蛋白SLC 1基因家族，是培养星形胶质细胞中谷氨酰胺的主要转运蛋白之一。除了谷氨酰胺和其他高亲和力底物丙氨酸、丝氨酸、半胱氨酸或苏氨酸外，ASCT 2还可以转运质子化谷氨酸。本研究阐明了ASCT 2在分化的人胎儿星形胶质细胞原代培养物中的底物依赖性运输。由8-溴-cAMP诱导的分化引起两种共定位和功能相关的星形胶质细胞蛋白-谷氨酸转运体GLAST（其是谷氨酸进入培养的星形胶质细胞的唯一高亲和力路由器）和谷氨酰胺合成酶（GS）（将至少一部分到达的谷氨酸转化为谷氨酰胺的胞质酶）的显著上调。为了区分这两种底物对ASCT 2的单独细胞内效应，在一些培养物中，使用siRNA沉默技术有效地敲低谷氨酰胺合成酶。在对照条件下，无论GS水平，几乎整个ASCT 2免疫反应性仅限于胞质溶胶。谷氨酰胺和丙氨酸。尽管程度不同。诱导ASCT 2从胞质区室到质膜的部分再分布。然而，在GS高表达的培养物中。微摩尔浓度的谷氨酸对ASCT 2运输的影响比该载体的优选底物更显著。相反。谷氨酸对缺乏GS的培养物中ASCT 2的分布没有影响。D-天冬氨酸是一种通过GLAST有效转运的代谢惰性底物，在所用的任何细胞培养物中均无影响。看来，细胞内谷氨酰胺产生的GS从谷氨酸，反过来。由GLAST提供。是比ASCT 2介导的细胞外底物易位更有效的ASCT 2运输至细胞表面的诱导剂。在较低的pH值（6.2-6.7），ASCT 2的细胞表面池显着大于生理pH值。此外，高浓度的谷氨酸，独立于GLAST或谷氨酸受体激活，诱导ASCT 2进一步到达质膜。ASCT 2的pH依赖性功能激活和ASCT 2介导的谷氨酸摄取可能在缺血性酸中毒或突触活性诱导的局部酸化过程中发挥重要作用。(C)2006爱思唯尔有限公司保留所有权利。
Neutral amino acid transporter ASCT2, together with high-affinity glutamate transporters, belongs to the SLC1 gene family of Na+-dependent solute carriers and is one of the major transporters of glutamine in cultured astrocytes. Besides glutamine and other high-affinity substrates-alanine, serine, cysteine or threonine, ASCT2 can also translocate protonated glutamate. The present study elucidated substrate-dependent trafficking of ASCT2 in differentiated primary Cultures of human fetal astrocytes. The differentiation induced by 8-bromo-cAMP caused dramatic up-regulation of two co-localized and functionally linked astroglial proteins-glutamate transporter GLAST, that is the only high-affinity router of glutamate into cultured astrocytes, and glutamine synthetase (GS), a cytosolic enzyme that converts at least a part of the arriving glutamate into glutamine. In order to distinguish individual intracellular effects of these two substrates on ASCT2, in some cultures glutamine synthetase was effectively knocked down using siRNA silencing technique. In control conditions, regardless of GS levels, almost the entire ASCT2 immunoreactivity was restricted to the cytosol. Both glutamine and alanine. though to different extents. induced partial redistribution of ASCT2 from the cytosolic compartment to the plasma membrane. However, in cultures with high GS expression. micromolar concentrations of glutamate exhibited more pronounced effect on ASCT2 trafficking than the preferred substrates of this carrier. In contrast. glutamate had no effect on ASCT2 distribution in cultures devoid of GS. D-Aspartate, a metabolically inert substrate effectively transported by GLAST had no effect in any cell culture utilized. It seems that intracellular glutamine produced by GS from glutamate that, in turn. is supplied by GLAST. is a more potent inducer of ASCT2 trafficking to the cell surface than the ASCT2-mediated translocation of extracellular substrates. At lower pH values (6.2-6.7), the cell surface pool of ASCT2 was significantly larger than at physiological pH. In addition, high concentrations of glutamate, independently from GLAST or glutamate receptor activation, induced further arrival of ASCT2 to the plasma membrane. The pH-dependent functional activation of ASCT2 and the ASCT2-mediated glutamate uptake may play important roles during ischemic acidosis or synaptic activity-induced local acidification. (C) 2006 Elsevier Ltd. All rights reserved.