Comparative pharmacology of the human NMDA-receptor subtypes R1-2A, R1-2B, R1-2C and R1-2D using an inducible expression system
Comparative pharmacology of the human NMDA-receptor subtypes R1-2A, R1-2B, R1-2C and R1-2D using an inducible expression system
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DOI：
10.1016/j.ejphar.2010.04.002
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发表时间：
2010-07-10
影响因子：
5
通讯作者：
Koller, Manuel
中科院分区：
文献类型：
--
作者：
Feuerbach, Dominik;Loetscher, Erika;Koller, Manuel
N-甲基-D-天冬氨酸(NMDA)受体在重组系统中功能表达后难以克服细胞毒性，这阻碍了其药理特性的研究。在这项研究中，使用了Milisterone诱导的NNMDA-R1亚单位的表达系统。这与NMDA-R2A、2B、2C和2D在不同细胞克隆中的结构性表达相结合。细胞系建立后，定量RT-PCR证实了NNMDA-R1亚基的可诱导性，并验证了NMDA-R2亚基在不同细胞克隆中的表达。功能反应的特征是通过离子通道的钙内流作为一个健壮的检测系统。刺激不同细胞系的N-甲基-D-天冬氨酸受体亚型导致钙瞬变逐渐升高，在S 30-160后达到高峰，之后缓慢下降。四种不同的NMDA受体亚型在相同的细胞背景中的表达使不同受体的直接药理学比较成为可能。谷氨酸在NMDA-R1-2D上显示出最高的效力。与谷氨酸相比，NMDA在所有亚型都显示出较低的效力，除NMDA-R1-2D外，NMDA是部分激动剂。本研究测试了20种拮抗剂，对谷氨酸引起的细胞内游离钙升高的抑制作用的药理学特征表明，每种受体亚型的拮抗剂效力有明显的等级顺序。这些数据表明，在相同的细胞背景下，评估受体刺激下的钙瞬变是比较作用于不同NMDA-R2受体的化合物的功能效应的有力工具。(C)2010爱思唯尔B.V.保留所有权利。
Pharmacological characterization of N-methyl-D-aspartate (NMDA) receptors has been hampered by the difficulty to outwit cytotoxicity after functional expression in recombinant systems. In this study a muristerone-inducible expression system for the NNMDA-R1 subunit was used. This was combined with constitutive expression of NMDA-R2A, 2B, 2C and 2D in different cell clones. After establishment of the cell lines, quantitative RT-PCR demonstrated the inducibility of the NNMDA-R1 subunit, and verified the expression of the NMDA-R2 subunits in the different cell clones. Functional responses were characterized using calcium influx through the ion channel as a robust assay system. Stimulation of the NMDA-receptor subtypes in the different cell lines led to calcium transients which were rising gradually, peaked after 30-160 s and declined thereafter very slowly. The expression of the four different NMDA-receptor subtypes in the same cellular background allowed a direct pharmacological comparison of the different receptors. Glutamate showed the highest potency at the NMDA-R1-2D. NMDA displayed at all subtypes a lower potency compared to glutamate and was a partial agonist except at the NMDA-R1-2D. 20 antagonists were tested in this study and the pharmacological characterization of the inhibition of glutamate-evoked elevation of intracellular free Ca(2+) revealed a distinct rank order of antagonist potency for each receptor subtype. These data illustrate that assessment of calcium transients upon receptor stimulation in the same cellular background is a powerful tool to compare the functional effects of compounds acting at the different NMDA-R2 receptors. (C) 2010 Elsevier B.V. All rights reserved.