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Ca++-Induced G Protein Coupled Receptor (GPCR) Activation Monitored via Aequorin Luminescence in the LMax Microplate Luminometer (MaxLine Application Note #42)

Jinfang Liao, M.D., Ph.D. and Evelyn McGown, Ph.D.
Molecular Devices Corporation, 8/00


INTRODUCTION
The photoprotein aequorin, a bioluminescent protein from the coelentratejellyfish Aequorea victorea, has proven to be highly sensitive to the trace amount of Ca++ in cells. The aequorin complex which contains the 22,000 MW apo-aequorinprotein, molecular oxygen, and the luminophore coelenterazine1,2 emits blue light (469 nm) upon binding calcium ions (Figure 1). The binding of Ca++ to aequorin induces a conformational change resulting in the oxidation ofcoelenterazine through an intramolecular reaction. This bioluminescent processpresents an advantage over Ca++-sensitive fluorescent dyes by being easilytargeted to specific cells and to subcellular compartments with appropriateregulatory elements and peptide signals3. Moreover, the aequorin complex doesnot require light excitation, thus eliminating autofluorescence, photobleachingand biological degradation problems. Its high affinity for Ca++ (Kd = 10 M) makes aequorin a good sensor in the biological range of intracellular Ca++ concentrations. The quick flash luminescence that is produced by aequorin canbe measured easily with the Lmax microplate luminometer equipped withautomated reagent injectors.

In a related previous application note4, we have described how to use Molecular Devices Lmax microplate luminometer to measure luciferase activity. Here, wedemonstrate how to use Lmax to detect receptor-activated Ca++ signals in cellsstably expressing apo-aequorin. In these cell-based assays, we were able toconstruct agonist concentration-response curves for the activation of a Gqprotein-coupled purinergic receptor expressed endogenously in CHO cells. Thus,the Lmax microplate luminometer can be used to study GPCR pharmacology.Furthermore, w
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