Recombinant aequorin as reporter of changes in intracellular calcium in mammalian cells

doi:10.1016/S0076-6879(00)27296-0

Methods in Enzymology

Volume 327, 2000, Pages 456-471

https://doi.org/10.1016/S0076-6879(00)27296-0 Get rights and content

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The photoprotein aequorin, from the coelenterate jellyfish Aequorea victoria, is a bioluminescent complex formed from the 21-kDa apoaequorin, the luminophore cofactor coelenterazine, and molecular oxygen. Apoaequorin is a 189-amino acid polypeptide chain containing three calcium-binding sites (EF-hand structures). After the binding of calcium to these sites, aequorin undergoes a conformational change, converting into an oxygenase. Aequorin has been used as a reporter of changes in intracellular calcium concentration in mammalian cells or Xenopus oocytes. In these experiments, loading of cells with aequorin has been involved microinjection of purified protein. Aequorin offers a highly sensitive method with which to assess changes in the level of intracellular calcium that offers significant time savings over the use of calcium-sensitive fluorescent dyes such as Fura-2 or Fluo-3. The detection of changes in intracellular calcium concentration using expressed aequorin does not require loading of the cells with dye, the extensive washing steps involved in the dye-loading procedure, the leaching of dye from the cells, or fluorescence quenching associated with the use of these dyes.

References (29)

BriniM. et al.
J. Biol. Chem.
(1995)
SheuY. et al.
Anal. Biochem.
(1993)
ButtonD. et al.
Cell Calcium
(1993)
RizzutoR. et al.
Methods Enzymol.
(1995)
BadmintonM. et al.
Exp. Cell Res.
(1995)
KendallJ. et al.
Anal. Biochem.
(1994)
NakahashiY. et al.
J. Biol. Chem.
(1997)
InouyeS. et al.
Anal. Biochem.
(1992)
BoieY. et al.
Eur. J. Pharmacol.
(1997)
WuD. et al.
J. Biol. Chem.
(1995)

MaedaA. et al.

Anal. Biochem.

(1996)

NorthR.A. et al.

Curr. Opin. Neurobiol.

(1997)

StablesJ. et al.

Anal. Biochem.

(1997)

SipmaH. et al.

Eur. J. Phannacol.

(1996)

Cited by (27)

Bioluminescence and kinetic aspects of double mutated aequorin variants
2018, International Journal of Biological Macromolecules
Citation Excerpt :
In the presence of Ca2+, even in trace amounts, conformational changes of aequorin will result in conversion of hydroperoxycoelenterazine (which is formed from coelenterazine and molecular oxygen) to coelenteramide along with release of CO2 [4,5]. Decarboxylation of coelenterazine leads to fast release of photons and subsequently emission of an instant flash of blue light (λmax = 469 nm) in less than 10 s [6–8]. Molecular biology techniques have been extensively used to improve the emission properties of existing photoproteins through production of mutated variants.
Aequorin as an old small calcium-sensitive photoprotein is a blue fluorescence protein which converts coelenterazine (a substrate) to coelenteramide with a flash type emission. The decay kinetics and emission properties of this protein can be changed using directed mutagenesis of crucial amino acid residue. In this work, we prepared three double mutants: Y⁸²F/W⁸⁶F, Y⁸²F/D¹⁵³G, and W⁸⁶F/D¹⁵³G. According to our results, it seems that presence of Y⁸²F mutation results in shift of emission to longer wavelengths while the W⁸⁶F mutation shifts the emission to shorter wavelengths. Furthermore, comparison of the variants for light half-life indicated decreased t_1/2 for the two variants of Y⁸²F/D¹⁵³G and W⁸⁶F/D¹⁵³G. But in compared to wild type aequorin, the Y⁸²F/W⁸⁶F variant displayed a 2-fold increase of light half-life. On the other hand, the thermostability properties of double mutants confirmed that only Y⁸²F/D¹⁵³G variant of apoaequorin is higher stability than others. Also, the single W⁸⁶F mutant reached the highest stability against thermal shock. Our data suggest that replacement of single or few point mutations in the binding pocket or active site of aequorin affects its bioluminescence and kinetic properties and so could be used for new reporter production of this photoprotein with the feasibility and limited substitutions.
Elevenin regulates the body color through a G protein-coupled receptor NlA42 in the brown planthopper Nilaparvata lugens
2018, General and Comparative Endocrinology
Citation Excerpt :
The selected polyclonal cells, HEK-NlA42 cells, were stored at −80 °C until use. Aequorin (AEQ) assays were performed essentially as described in the methods of Button and Brownstein (1993) and Stables et al. (2000). PcDNA3-mtAEQ, containing mitochondria-targeted apoaequorin (mtAEQ), was transiently transfected into HEK-NlA42 cells and then treated with 5 μM coelenterazine h (Wako, Tokyo, Japan).
The neuropeptide elevenin and similar neuropeptide precursors are common in some invertebrates but their physiological function in most species has not been explored. The brown planthopper, Nilaparvata lugens (Stål) has an elevenin-like peptide and a G protein-coupled receptor (GPCR) NlA42 that is homologous to the elevenin receptor of the annelid Platynereis dumerilii. RNA interference (RNAi)-mediated knockdown of either Nl-elevenin or the NlA42 gene resulted in cuticle melanization. Ion transport peptide (ITP) also induces melanization, but unlike ITP, knockdown of NlElevenin and NlA42 did not have any effect on wing expansion or activity after eclosion. In wild condition macropterous individuals show a darker body color when compared with brachypterous individuals, but RNAi experiments suggest that insulin-signaling and Nl-elevenin signaling regulate wing morph and body color independently. NlElevenin was predominantly expressed in the brain while NlA42 was highly expressed in the abdominal integument and brain. A signal Calcium assays using aequorin indicated that NlA42 heterologously expressed in HEK293 cells exhibited responses to synthetic Nl-elevenin peptide from concentrations as low as 10⁻⁹ M. These results suggest that neuropeptide Nl-elevenin is involved in the regulation of melanization through its receptor NlA42. This is the first report of a physiological function for elevenin-like peptides in insects.
Pharmacology, Signaling and Physiological Relevance of the G Protein-coupled Receptor 55
2011, Advances in Pharmacology
Citation Excerpt :
Mammalian cell experiments were performed in a GPR55 stably expressing recombinant HEK293aeq cell line, which also carries the apoaequorin gene of the jellyfish Aequorea victoria. This system allows bioluminescent measurement of intracellular Ca2+ concentrations (Stables et al., 2000). A series of selective small-molecule ligands all containing a benzoylpiperazine structure were identified as putative GPR55 agonists.
According to The European Monitoring Centre for Drugs and Drug Addiction (EMCDDA), ∼70 million European adults have consumed cannabis on at least one occasion. Cannabis consumption leads to a variety of psychoactive effects due to the presence of the constituent Δ⁹-tetrahydrocannabinol (Δ⁹-THC). Δ⁹-THC interacts with the endocannabinoid system (ECS), which consists of the seven transmembrane spanning (7TM)/G protein-coupled receptors (GPCRs) CB₁ and CB₂, their respective ligands (endocannabinoids), and enzymes involved in their biosynthesis and degradation. This system plays a critical role in many physiological processes such as learning and memory, appetite control, pain sensation, motor coordination, lipogenesis, modulation of immune response, and the regulation of bone mass. Therefore, a huge effort has been spent trying to fully elucidate the composition and function of the ECS. The G protein-coupled receptor 55 (GPR55) was recently proposed as a novel component of this system; however, its classification as a cannabinoid receptor has been significantly hampered by its complex pharmacology, signaling, and cellular function. GPR55 is phylogenetically distinct from the traditional cannabinoid receptors, but in some experimental paradigms, it is activated by endocannabinoids, phytocannabinoids, and synthetic cannabinoid ligands. However, the most potent compound appears to be a lysophospholipid known as lysophosphatidylinositol (LPI). Here, we provide a comprehensive evaluation of the current pharmacology and signaling of GPR55 and review the proposed role of this receptor in a number of physiological and pathophysiological processes.
Fluorescence- and luminescence-based methods for the determination of affinity and activity of neuropeptide Y<inf>2</inf> receptor ligands
2006, European Journal of Pharmacology
With respect to the discovery and characterization of neuropeptide Y₂ receptor ligands as pharmacological tools or potential drugs, fluorescence- and luminescence-based assays were developed to determine both the affinity and the activity of receptor agonists and antagonists. A flow cytometric binding assay is described for the hY₂ receptor stably expressed in CHO cells using cy5-labeled porcine neuropeptide Y and compared with a radioligand binding assay. Binding of the fluorescent ligand was visualized by confocal microscopy. Stable co-transfection with the chimeric G protein Gq_i5 enabled the establishment of a spectrofluorimetric fura-2 and a flow cytometric fluo-4 calcium assay. Further stable expression of apoaequorin targeted to the mitochondria allowed the establishment of an aequorin assay which could be performed in the 96-well format. The shape of the concentration–response curves of porcine neuropeptide Y in the presence of the Y₂-selective receptor antagonist BIIE0246, characteristic of either competitive or insurmountable antagonism, depended on the period of incubation with the cells. Functional data of Y₂ receptor agonists and antagonists determined in the fluorescence- and luminescence-based assays were in good agreement.
Optical probing of neuronal circuit dynamics: Genetically encoded versus classical fluorescent sensors
2006, Trends in Neurosciences
During the past few decades, optical methods for imaging activity in networks composed of thousands of neurons have been developed. These techniques rely mainly on organic-chemistry-based dyes as indicators of Ca²⁺ and membrane potential. However, recently a new generation of probes, genetically encoded fluorescent protein sensors, has emerged for use by physiologists studying the operation of neuronal circuits. We critically review the development of these new probes, and analyze objectives and experimental conditions in which classical probes are likely to prevail and where the fluorescent protein sensors will open paths to previously unexplored territories of functional neuroimaging.
Structure, function and mode of action of select arthropod neuropeptides
2006, Studies in Natural Products Chemistry
Citation Excerpt :
The molecular oxygen oxidizes coelenterazine and the emission of a blue light is one of the products formed [15]. Thus, in the bioassay when the agonist binds to the G protein-coupled receptor, the G protein is stimulated and results in an intracellular increase of Ca2 +; the latter combines with apoaequorin and the protein undergoes a conformational change into an oxygenase, coelenterazine is oxidized and a blue light is emitted and measured [15]. The electrophysiologically based receptor bioassay relies on measuring a change in the membrane potential of cells when a ligand binds to the receptor of interest.
This overview summarizes important features of the majority of neuropeptide families that occur in two species-rich and widely-radiated arthropod taxa, the crustaceans and the insects. The neuropeptides may act as true neurohormones, which are released into the circulation, or as local neurotransmitter and/or neuromodulator. By comparing the primary structures of members of peptide families, the biosynthesis (including preprohormone structure and the peptidergic control of release), the structures of the receptors and transduction of the message via second messenger systems, the inactivation and the multiple functions of selected neuropeptides, we want to draw the reader’s attention to the following main conclusions: 1) neuropeptides are important physiological regulators in arthropods; 2) neuropeptides can be structurally and functionally highly conserved in major arthropod groups (for example, proctolin, crustacean cardioactive peptide), or where peptide isoforms exist, there may be different scenarios: - (i) in the case of identical isoforms in insects and crustaceans, the peptide function may have changed in the two taxa (for example, red pigment-concentrating hormone affects pigmentation in crustaceans but mobilizes lipids in insects), - (ii) the isoforms are different in the two taxa and may have the same effect (for example, the ion-transporting peptide in insects and the crustacean hyperglycaemic hormone in crustaceans both play a role in osmoregulation), - (iii) the structurally different isoforms have different functions in the two taxa (for example, pigment-dispersing hormone affects pigmentation in crustaceans, whereas pigment-dispersing factor affects circadian rhythmicity in insects.

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Recombinant aequorin as reporter of changes in intracellular calcium in mammalian cells

Publisher Summary

J. Biol. Chem.

Anal. Biochem.

Cell Calcium

Methods Enzymol.

Exp. Cell Res.

Anal. Biochem.

J. Biol. Chem.

Anal. Biochem.

Eur. J. Pharmacol.

J. Biol. Chem.

Anal. Biochem.

Curr. Opin. Neurobiol.

Anal. Biochem.

Eur. J. Phannacol.