Functional and pharmacological differences between recombinant N-methyl-D-aspartate receptors
Functional and pharmacological differences between recombinant N-methyl-D-aspartate receptors
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DOI：
10.1152/jn.1998.79.2.555
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发表时间：
1998-02-01
影响因子：
2.5
通讯作者：
Grayson, DR
中科院分区：
文献类型：
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作者：
Vicini, S;Wang, JF;Grayson, DR
将n -甲基- d -天冬氨酸（NMDA）受体瞬时转染到哺乳动物HEK-293细胞中，用亚单位特异性抗体和电生理记录对其进行了表征。研究了快速l -谷氨酸脉冲对细胞失活时间的影响，并研究了从表达重组NR1亚基与NR2A、NR2B、NR2C或NR2D NMDA受体亚基结合的细胞中切除的外膜斑块。利用共表达的维多利亚海鞘绿灯蛋白发出的荧光预先鉴定转染的细胞。NR1/NR2A通道产生的电流比NR1/NR2B或NR1/NR2C通道产生的电流表现出双指数失活时间过程。然而，在每次共转染中观察到很大的衰减变异性，这表明除了亚基组成之外的机制也可能调节失活时间过程。NR1/NR2D通道显示缓慢失活电流。通过共转染NR1亚基的五种不同剪接变体，每个NR2A亚基获得的受体通道失活速度快，且具有可同性。此外，NR1/NR2B通道脱敏后的恢复速度比NR1/NR2A通道慢。在以可变比例共转染NR1/NR2A/NR2B cdna的细胞中，对l -谷氨酸的短暂应用反应的平均失活时间过程介于NR1/NR2A和NR1/NR2B通道之间。尽管免疫细胞化学证据表明，大多数细胞在三重转染中被所有质粒共转染，但我们的实验条件不允许严格控制NMDA受体亚基的表达。结果表明，许多细胞具有NR1/NR2A和NR1/NR2B亚基异构体的失活时间和氟哌啶醇敏感性。我们还推测，少数细胞对l -谷氨酸的短暂应用有反应，其特征是失活时间缓慢，氟哌啶醇敏感性降低，因此NR1/NR2A/NR2B亚基的sam受体聚集可能形成通道。
N-methyl-D-aspartic acid(NMDA) receptors transiently transfected into mammalian HEK-293 cells were characterized with subunit-specific antibodies and electrophysiological recordings. Deactivation time course recorded in response to fast L-glutamate pulses were studied in isolated and lifted cells, as well as in outside-out membrane patches excised from cells expressing recombinant NR1 subunits in combination with the NR2A, NR2B, NR2C, or NR2D NMDA receptor subunits. Transfected cells were preidentified by the fluorescence emitted from the coexpressed Aequorea victoria jellyfish Green Lantern protein. Currents generated by NR1/NR2A channels displayed double exponential deactivation time course being faster than that in NR1/NR2B or NR1/NR2C channels. However, a large decay variability was observed within each cotransfection, suggesting that mechanisms additional to subunit composition may also regulate deactivation time course. NR1/NR2D channels displayed slowly deactivating currents. Channel deactivation was fast and comparable among receptors obtained by cotransfecting five distinct spliced variants of the NR1 subunit, each with the NR2A subunit. Additionally, recovery from desensitization was slower for NR1/NR2B than for NR1/NR2A channels. The average deactivation time course of responses to brief L-glutamate applications in cells where NR1/NR2A/NR2B cDNAs were cotransfected at variable ratio was intermediate between those of the NR1/NR2A and NR1/NR2B channels. Although immunocytochemical evidence indicates that the majority of cells are cotransfected by all plasmids in triple transfection, our experimental condition did not allow for a tight control of the expression of NMDA receptor subunits. This produced the result that many cells were characterized by deactivation time course and haloperidol sensitivities of separate NR1/NR2A and NR1/NR2B subunit heteromers. We also speculate on the possible formation of channels resulting from the coassembly in the sam-receptor of NR1/NR2A/NR2B subunits from a minority of cells that gave responses to brief application of L-glutamate characterized by slow deactivation time course and decreased haloperidol sensitivity.