Binding and transport of [3H] (2S,4R)4-methylglutamate, a new ligand for glutamate transporters, demonstrate labeling of EAAT1 in cultured murine astrocytes
Binding and transport of [3H] (2S,4R)4-methylglutamate, a new ligand for glutamate transporters, demonstrate labeling of EAAT1 in cultured murine astrocytes
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DOI：
10.1002/jnr.20013
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发表时间：
2004-03-15
影响因子：
4.2
通讯作者：
O'Shea, RD
中科院分区：
文献类型：
--
作者：
Apricò, K;Beart, PM;O'Shea, RD
L-谷氨酸转运蛋白（兴奋性氨基酸转运蛋白; EAAT），定位于星形胶质细胞，密切参与脑内的中间代谢。由于（2S，4 R）-4-甲基谷氨酸（4 MG）对胶质细胞EAAT具有亲和力，我们采用[H-3] 4 MG来确定培养的小鼠星形胶质细胞中EAAT的特征，并描述分析EAAT功能的新方法。4 ℃时[H-3] 4 MG在星形胶质细胞膜上的特异性结合占总结合的90%。结合迅速（表观t（1/2）与7 min相似）且可饱和。饱和度和Scatchard分析表明单一结合位点（n（H）= 0.8），K-d为6.0 +/- 1.5 μ M，B-max = 9.7 +/- 2.9 pmol/mg蛋白。[H-3] 4 MG与星形胶质细胞匀浆的结合是Na+依赖性的，并被K+抑制。作用于EAAT的化合物，如L-谷氨酸（Glu）、D-天冬氨酸（D-Asp）、L-（2S，3S，4 R）-2（羧基环丙基）甘氨酸和L-反式-吡咯烷-2，4-二羧酸酯，将结合置换至非特异性水平。L-丝氨酸-O-硫酸盐，一种EAAT 1偏好配体，完全取代[H-3] 4 MG的结合。相比之下，对EAAT 2、L-苏型-3-甲基谷氨酸盐、二氢红藻氨酸盐和红藻氨酸盐具有优先亲和力的抑制剂是相对无效的结合置换剂。Glu受体的激动剂和拮抗剂未能显著抑制[H-3] 4 MG结合。用[H-3]D-Asp进行的研究进一步证实了[H-3] 4 MG与EAAT结合的证据。这些数据与Western印迹分析一致，表明EAAT 1而不是EAAT 2的丰富表达。[H-3] 4 MG也通过钠和温度敏感性过程（K-m为146 +/- 24 μ M，V-max = 336 +/- 27 nmol/mg蛋白/min）快速积累（表观t（1/2）类似于4 min）到整个星形胶质细胞中，其具有EAAT 1样药理学特征。这些发现证实了4 MG是EAAT 1的底物，并且使用[H-3] 4 MG开发的结合测定可用于包括培养的星形胶质细胞在内的各种制剂。(C)2004 Wiley-Liss，Inc.
Transporters for L-glutamate (excitatory amino acid transporters; EAATs), localized to astrocytes, are involved intimately in intermediary metabolism within the brain. Because (2S,4R)-4-methylglutamate (4MG) has affinity for glial EAATs, we employed [H-3]4MG to define the characteristics of EAATs in cultured murine astrocytes and describe new approaches to analyze EAAT function. Specific binding of [H-3]4MG in astrocytic membranes at 4degreesC represented 90% of total binding. Binding was rapid (apparent t(1/2)similar to7 min) and saturable. Saturation and Scatchard analyses indicated a single binding site (n(H) = 0.8) with a K-d of 6.0 +/- 1.5 muM and B-max = 9.7 +/- 2.9 pmol/mg protein. Binding of [H-3]4MG to astrocytic homogenates was Na+-dependent and inhibited by K+. Compounds acting at EAATs, such as L-glutamate (Glu), D-aspartate (D-Asp), L-(2S,3S,4R)-2(carboxycyclopropyl)glycine and L-trans-pyrrolidine-2,4-dicarboxylate displaced binding to nonspecific levels. L-Serine-O-sulphate, an EAAT1 -preferring ligand, fully displaced binding of [H-3]4MG. In contrast, inhibitors having preferential affinity for EAAT2, L-threo-3-methylglutamate, dihydrokainate, and kainate, were relatively ineffective binding displacers. Agonists and antagonists for Glu receptors failed to significantly inhibit [H-3]4MG binding. Studies with [H-3]D-Asp reinforced evidence that [H-3]4MG was binding to EAATs. These data were consistent with Western blot analyses, which indicated abundant expression of EAAT1 but not EAAT2. [H-3]4MG was also accumulated rapidly (apparent t(1/2) similar to4 min) into whole astrocytes by a sodium- and temperature-sensitive process (K-m of 146 +/- 24 muM, V-max = 336 +/- 27 nmol/mg protein/min), which possessed an EAAT1-like pharmacologic profile. These findings confirm that 4MG is a substrate for EAAT1 and that the binding assay developed using [H-3]4MG can be utilized in various preparations including cultured astrocytes. (C) 2004 Wiley-Liss, Inc.