Recombinant aequorin as a reporter for receptor-mediated changes of intracellular Ca2+-levels in Drosophila S2 cells
Recombinant aequorin as a reporter for receptor-mediated changes of intracellular Ca2+-levels in Drosophila S2 cells
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重组水母发光蛋白作为果蝇 S2 细胞受体介导的细胞内 Ca2+ 水平变化的报告基因
DOI：
10.1007/s10158-001-0013-2
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发表时间：
2002
影响因子：
--
通讯作者：
J. Vanden Broeck
中科院分区：
文献类型：
--
作者：
H. Torfs;J. Poels;M. Detheux;V. Dupriez;T. Van Loy;L. Vercammen;G. Vassart;M. Parmentier;J. Vanden Broeck
抽象。利用生物发光的Ca 2+敏感报告蛋白水母发光蛋白（aequorin）建立了一种基于昆虫细胞的功能检测系统，用于监测受体介导的细胞内Ca 2+浓度变化。果蝇Schneider 2（S2）细胞的基因工程稳定表达脱辅基水母发光蛋白和昆虫速激肽相关肽受体，STKR。Lom-TK III是一种STKR激动剂，可在这些S2-STKR-Aeq细胞中引发浓度依赖性生物发光反应。通过水母发光蛋白检测的钙效应的EC 50值似乎与通过Fura-2（一种荧光Ca 2+指示剂）测量的EC 50值几乎相同。此外，这种基于水母发光蛋白的方法也用于研究受体拮抗剂。三种有效的P物质拮抗剂Spantide I、II和III对Lom-TK III刺激的S2-STKR-Aeq细胞的作用的实验分析表明，这些化合物以浓度依赖性方式拮抗STKR介导的反应。抑菌活性大小顺序为spantide Ⅲ> spantide Ⅱ> spantide Ⅰ。
Abstract. The bioluminescent Ca2+-sensitive reporter protein, aequorin, was employed to develop an insect cell-based functional assay system for monitoring receptor-mediated changes of intracellular Ca2+-concentrations. Drosophila Schneider 2 (S2) cells were genetically engineered to stably express both apoaequorin and the insect tachykinin-related peptide receptor, STKR. Lom-TK III, an STKR agonist, was shown to elicit concentration-dependent bioluminescent responses in these S2-STKR-Aeq cells. The EC50 value for the calcium effect detected by means of aequorin appeared to be nearly identical to the one that was measured by means of Fura-2, a fluorescent Ca2+-indicator. In addition, this aequorin-based method was also utilised to study receptor antagonists. Experimental analysis of the effects exerted by spantide I, II and III, three potent substance P antagonists, on Lom-TK III-stimulated S2-STKR-Aeq cells showed that these compounds antagonise STKR-mediated responses in a concentration-dependent manner. The rank order of inhibitory potencies was spantide III > spantide II > spantide I.