( Jinfang Liao M.D. Ph.D. and Evel...)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 coelenterazine^1,2 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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