AEQUORIN-EXPRESSING MAMMALIAN-CELL LINES USED TO REPORT CA-2+ MOBILIZATION
AEQUORIN-EXPRESSING MAMMALIAN-CELL LINES USED TO REPORT CA-2+ MOBILIZATION
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DOI：
10.1016/0143-4160(93)90091-j
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发表时间：
1993-10-01
期刊：
影响因子：
4
通讯作者：
BROWNSTEIN, M
中科院分区：
文献类型：
--
作者：
BUTTON, D;BROWNSTEIN, M
稳定表达水母水母发光蛋白的哺乳动物细胞已被用于报告细胞表面受体激活Ca 2+动员。在CHO-K1和293细胞中，水母发光蛋白cDNA（pAEQ）的表达由巨细胞病毒（CMV）启动子驱动。获得表达高水平的脱辅基水母发光蛋白的克隆分离物，脱辅基水母发光蛋白的Ca 2+依赖性发光是通过用腔肠动物白蛋白、腔肠素处理活细胞产生的。水母发光蛋白在COS细胞中的瞬时表达导致发光蛋白的更大丰度。水母发光蛋白从毛地黄皂苷透化的细胞中以与乳酸脱氢酶（LDH）相同的程度和速率丢失，表明指示剂的细胞溶质位置。用内源性受体激动剂处理的水母发光蛋白表达细胞用于发光测量，以证明报告细胞系提供了测定胞质Ca 2+离子浓度变化的高度灵敏和稳健的方法。还进行了水母发光蛋白报告细胞中P物质受体的瞬时共表达，以证明使用这种方便和灵敏的测定系统用于大规模筛选活化细胞表面受体的配体的可行性，所述配体与细胞内Ca 2+的增加偶联。
Mammalian cells that stably express jellyfish aequorin have been used to report activation of Ca2+ mobilization by cell-surface receptors. Expression of aequorin cDNA (pAEQ) was driven by the cytomegalovirus (CMV) promotor in CHO-K1 and 293 cells. Clonal isolates were obtained which express high levels of apo-aequorin protein, the Ca2+-dependent luminescence of which is generated by treatment of living cells with the coelenterate luciferin, coelenterazine. Transient expression of aequorin in COS cells results in even greater abundance of luminescent protein. Aequorin protein is lost from digitonin-permeabilized cells to the same extent and at the same rate as lactate dehydrogenase (LDH), indicating cytosolic location of the indicator. Aequorin expressing cells treated with agonists of endogenous receptors were used in luminescence measurements to demonstrate that the reporter lines offer a highly sensitive and robust means of assaying changes in the concentration of cytosolic Ca2+ ion. Transient co-expression of the substance P receptor in aequorin reporter cells was also performed to demonstrate the feasibility of using this convenient and sensitive assay system for large scale screening of ligands that activate cell surface receptors coupled to increases in intracellular Ca2+.