Aequorin: superior choice for GPCR calcium signalling

In the realm of cellular signalling, GPCRs are pivotal in transmitting extracellular stimuli to intracellular responses, often involving calcium ions (Ca²⁺) as secondary messengers. To effectively study these intricate pathways, a precise reporter system is essential. Aequorin, a photoprotein originally derived from the jellyfish Aequorea victoria, has emerged as a superior reporter for GPCR Ca²+ signalling.

A primary advantage of Aequorin is its high sensitivity to Ca²+. Upon binding with Ca²+, Aequorin undergoes a conformational change, leading to the oxidation of its coelenterazine substrate and the emission of blue light. This bioluminescent reaction is highly specific to Ca²+, ensuring minimal interference from other ions or cellular components. Because background noise is greatly reduced compared to other approaches that use fluorescent dyes, the use of Aequorin offers a larger assay window and allows for more accurate real-time monitoring of Ca²+ fluxes within living cells, providing invaluable insights into GPCR-mediated signalling events.

Another critical benefit of Aequorin is its ability to be expressed in various subcellular compartments, such as mitochondria, using a dedicated signal peptide sequence. Ca2+ fluxes are more intense in the mitochondria, upon cytosolic Ca2+ increase. This improves the sensitivity of mitochondria-targeted Aequorin reporters. Moreover, progressive mutagenesis has allowed the generation of variants beyond wild-type Aequorin with improved stability, light emission, Ca2+ affinity.

Illuminating Cell Communication
Aequorin’s versatility in various experimental setups is noteworthy, including single-cell analyses, high-throughput screening, and even whole-organism studies. This adaptability makes Aequorin an ideal choice for a wide range of applications, from basic research to drug discovery. Furthermore, the bioluminescent signal generated by Aequorin is compatible with most standard laboratory equipment which allows EuroscreenFast to facilitate seamless integration into existing workflows.

The resolution offered by Aequorin is another advantage. GPCR signalling events occur on rapid timescales, necessitating reporters that can keep pace with these dynamic changes. Aequorin’s fast response to Ca2+ binding allows for the real-time tracking of transient Ca²⁺ signals, capturing the nuances of GPCR-mediated processes that might be missed by slower-responding indicators.

Aequorin’s bioluminescence is also advantageous in reducing background noise, a common issue with fluorescence-based indicators. Since bioluminescence does not require external excitation light, it minimizes autofluorescence and photobleaching, thereby enhancing signal-to-noise ratio and improving the accuracy of measurements. This feature is especially beneficial in complex biological environments where autofluorescence can obscure subtle changes in Ca2+ levels.
The application of Aequorin in GPCR Ca2+ signalling has already yielded significant insights. For instance, it has been instrumental in elucidating the role of Ca2+ in neurotransmitter release, hormone secretion, and sensory perception. In EuroscreenFast’s hands, Aequorin has helped advance understanding of cellular communication and even led to the deorphanisation of a number of GPCRs.

The advantages of Aequorin as a reporter for GPCR Ca2+ signalling are manifold. High sensitivity, specificity, sub-cellular compartment targeting, versatility, and superior temporal resolution make it an invaluable tool. By illuminating the intricate dance of Ca2+ ions within cells, Aequorin paves the way for groundbreaking discoveries in cellular signalling and beyond. As EuroscreenFast continues to explore the depths of cellular communication, Aequorin will undoubtedly remain a beacon of clarity and precision in the study of GPCR Ca2+ signalling.

Author: Dr Laurent Meeus, Chief Scientific Officer & General Manager, EuroscreenFast

This article was originally published in European Biotechnology Magazine Autumn 2024.