Aequorin-expressing mammalian cell lines used to report Ca2+ mobilization.
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) promoter in CHO-K1 and 293 cells. Clonal isolates were obtained which express high levels of apo-aequorin protein, the Ca(2+)-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+.
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Laboratory of Cell Biology, NIMH, NIH, Bethesda, Maryland.
Brownstein
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D000331:Aequorin / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D006224:Cricetinae; D005240:Feasibility Studies; D005786:Gene Expression Regulation; D006801:Humans; D007093:Imidazoles; D007668:Kidney / Q000166:cytology; D008024:Ligands; D008163:Luminescent Measurements; D011719:Pyrazines; D013373:Substance P / Q000378:metabolism*; D014162:Transfection
D007093:Imidazoles; D008024:Ligands; D011719:Pyrazines; D013373:Substance P; C017144:coelenterazine; D000331:Aequorin; D002118:Calcium