Aequorin-Based Functional Assays for G-Protein-Coupled Receptors, Ion Channels, and Tyrosine Kinase Receptors

Abstract

Aequorin is a photoprotein originating from jellyfish, whose luminescent activity is dependent on the concentration of calcium ions. Due to the high sensitivity and low background linked to luminescent assays, as well as to its absence of toxicity and its large linear dynamic range, aequorin has been used as an intracellular calcium indicator since its discovery in the early 1960s. The first applications of aequorin involved its microinjection in cells. The cloning of its gene in 1985 opened the way to the stable expression of aequorin in cell lines or even entire organisms. Here we present the validation of aequorin as a functional assay for the screening of G-protein-coupled receptors, ion channels, and tyrosine kinase receptors, as well as for their pharmacological characterization in agonist and antagonist detection assays. We optimized our cell suspension-based assay and determined that the most sensitive assay was performed at room temperature, with mitochondrially expressed aequorin and using coelenterazine derivative h for reconstitution of aequorin. The robustness of the assay and the current availability of luminometers with integrated injectors allow aequorin to fit perfectly with high throughput functional assays requirements.

Interchange of aequorin and obelin bioluminescence color is determined by substitution of one active site residue of each photoprotein

“…In each case, the bioluminescence and fluorescence spectral properties were affected as would be expected based on the suggested proton‐relay mechanism [16]. In addition, we also show that these mutants have good stability and activity, and similar to that reported for aequorin itself [25–27] can be expressed in mammalian cells thereby demonstrating their potential for monitoring multiple events in cells, such as the simultaneous expression of different genes or the measurement of intracellular calcium in different cell compartments, for example.…”

“…In addition to direct measurement of [Ca 2+ ], photoproteins can be used in various indirect cell based assays [27]. For instance, if the binding of some agonist with a membrane receptor results in a stimulation of phospholipase C and therefore in activation of the inositol‐1,4,5‐trisphosphate (InsP 3 )/Ca 2+ signaling pathways [35], the “built in” photoprotein can be used for monitoring the activation of this membrane receptor.…”

5F‐MDMB‐PICA metabolite identification and cannabinoid receptor activity

“…This method is based on the activation of the calcium sensitive apoaequorin/aequorin system where coelenterazine is converted by the aequorin upon activation. After the G‐protein activation, the second messenger phospholipase C is activated, with succeeding diacylglycerol and inositoltriphosphate production and intracellular calcium ions release . The calcium release activates the apoaequorin/aequorin system and with coelenterazine as substrate luminescence is produced.…”

An Overview of High Throughput Screening at G Protein Coupled Receptors

“…Some compounds in the screening library may modulate intracellular calcium levels by means other than binding to the receptor; or may alter dye fluorescence, resulting in false positive or negative hits. Consequently, HTS platforms using the calcium sensitive bioluminescent protein, aequorin, have been developed [62,63]. The approach utilizes the calcium ion binding property of the protein, causing in oxidation of the substrate, coelenterazine, with concomitant light emission.…”