Inducible expression and pharmacology of the human excitatory amino acid transporter 2 subtype of L‐glutamate transporter
Inducible expression and pharmacology of the human excitatory amino acid transporter 2 subtype of L‐glutamate transporter
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L-谷氨酸转运蛋白人兴奋性氨基酸转运蛋白2亚型的诱导表达和药理学
DOI：
10.1038/sj.bjp.0702945
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发表时间：
1999
影响因子：
7.3
通讯作者：
H. McIlvain
中科院分区：
文献类型：
--
作者：
J. Dunlop;Z. Lou;Y. Zhang;H. McIlvain
在这项研究中，我们研究了利用蜕皮激素诱导的哺乳动物表达系统(Invitgen)来调节HEK293细胞中主要的L-谷氨酸转运体EAAT2(兴奋性氨基酸转运体)的表达。用稳定转化调控载体pVgRXR的HEK 293细胞(ECR 293细胞)，用可诱导载体Pind转染人EAAT2基因，筛选出HEK/EAAT2克隆进行进一步鉴定。在ECR293细胞中观察到依赖于Na+的L-谷氨酸摄取活性(3.2pmoL −1 mg−1)，而在未诱导的HEKEAT2细胞中，这一活性增加了近2倍，表明在没有外源诱导剂的情况下，基础EAAT2活性水平很低。蜕皮激素类似物Ponasterone A(10 μM作用24 h)可使HEK/EAT2细胞的Na+依赖活性增加10倍。L对谷氨酸的摄取遵循一级米氏动力学，饱和摄取数据的Eadie-Hofstee变换得到的动力学参数为：Km52.7±7.5 μM，Vmax 3.8±0.9 min−1 mg−1蛋白。用一系列L-谷氨酸转运抑制剂表征了EAT2亚型的药理学特征，其抑制效力的等级顺序与先前所述的大鼠同系物GLT-1和大鼠皮质突触体内制剂的等级顺序相似。在诱导的HEK/EAAT2细胞中加入EAT2调节剂花生四烯酸后，L-谷氨酸转运能力增强(30 μM时对照为15 5±5%)。这项研究表明，EAAT2的表达可以通过蜕皮激素诱导的哺乳动物表达系统在哺乳动物细胞系中进行调节。
In this study we have examined the use of the ecdysone‐inducible mammalian expression system (Invitrogen) for the regulation of expression of the predominant L‐glutamate transporter EAAT2 (Excitatory Amino Acid Transporter) in HEK 293 cells. HEK 293 cells which were stably transformed with the regulatory vector pVgRXR (EcR 293 cells) were used for transfection of the human EAAT2 cDNA using the inducible vector pIND and a clone designated HEK/EAAT2 was selected for further characterization. Na+‐dependent L‐glutamate uptake activity (3.2 pmol min−1 mg−1) was observed in EcR 293 cells and this was increased approximately 2 fold in the uninduced HEK/EAAT2 cells, indicating a low level of basal EAAT2 activity in the absence of exogenous inducing agent. Exposure of HEK/EAAT2 cells to the ecdysone analogue Ponasterone A (10 μM for 24 h) resulted in a 10 fold increase in the Na+‐dependent activity. L‐glutamate uptake into induced HEK/EAAT2 cells followed first‐order Michaelis‐Menten kinetics and Eadie‐Hofstee transformation of the saturable uptake data produced estimates of kinetic parameters as follows; Km 52.7±7.5 μM, Vmax 3.8±0.9 nmol min−1 mg−1 protein. The pharmacological profile of the EAAT2 subtype was characterized using a series of L‐glutamate transport inhibitors and the rank order of inhibitory potency was similar to that described previously for the rat homologue GLT‐1 and in synaptosomal preparations from rat cortex. Addition of the EAAT2 modulator arachidonic acid resulted in an enhancement (155±5% control in the presence of 30 μM) of the L‐glutamate transport capacity in the induced HEK/EAAT2 cells. This study demonstrates that the expression of EAAT2 can be regulated in a mammalian cell line using the ecdysone‐inducible mammalian expression system.
影响因子：
5.3
作者：
Figiel, M;Engele, J
通讯作者：
Engele, J
影响因子：
3.7
作者：
Conti, F;DeBiasi, S;Melone, M
通讯作者：
Melone, M