Dunlop J
Wyeth Neuroscience, Wyeth-Ayerst Research, Princeton, New Jersey 08543, USA.
J Neurosci Res. 2001 Nov 1;66(3):482-6. doi: 10.1002/jnr.1239.
A stable cell line expressing the predominant brain glutamate transporter EAAT2 was used for the characterization of substrate exchange as a biochemical index for discriminating between substrate and non-substrate inhibitors of the cloned EAAT2 transporter. Addition of 1 mM unlabeled D-aspartate to cells equilibrated with [3H]D-aspartate produced a time-dependent depletion of the [3H] label retained by the cells. L-Aspartate, L-glutamate and L-cysteate produced an equivalent degree of [3H] exchange to that observed with D-aspartate, although the non-substrate EAAT2 inhibitor dihydrokainate and D-glutamate, which does not interact with the substrate binding site, failed to stimulate [3H]D-aspartate exchange. Estimation of EC50 values for the stimulation of [3H] exchange by D-aspartate, L-glutamate and L-trans-2,4-pyrollidine carboxylate (trans-PDC) produced values that were in excellent agreement with the corresponding IC50 values for the same compounds to inhibit EAAT2 uptake. Moreover, trans-PDC was found to produce a lower maximal exchange than that observed with D-aspartate, consistent with the known partial EAAT2 substrate activity of trans-PDC. The estimate of drug induced [3H] efflux with the cloned EAAT2 transporter represents a convenient biochemical assay for the discrimination of substrate and non-substrate inhibitors of the EAAT2 subtype.
使用表达主要脑谷氨酸转运体EAAT2的稳定细胞系来表征底物交换，作为区分克隆的EAAT2转运体的底物抑制剂和非底物抑制剂的生化指标。向用[3H]天冬氨酸平衡的细胞中添加1 mM未标记的D-天冬氨酸，会导致细胞保留的[3H]标记随时间减少。L-天冬氨酸、L-谷氨酸和L-半胱氨酸产生的[3H]交换程度与D-天冬氨酸相当，尽管非底物EAAT2抑制剂二氢海因酸和不与底物结合位点相互作用的D-谷氨酸未能刺激[3H]天冬氨酸交换。对D-天冬氨酸、L-谷氨酸和L-反式-2,4-吡咯烷羧酸（反式-PDC）刺激[3H]交换的EC50值的估计，与相同化合物抑制EAAT2摄取的相应IC50值非常一致。此外，发现反式-PDC产生的最大交换低于D-天冬氨酸，这与反式-PDC已知的部分EAAT2底物活性一致。用克隆的EAAT2转运体估计药物诱导的[3H]流出，代表了一种方便的生化测定方法，用于区分EAAT2亚型的底物抑制剂和非底物抑制剂。
