Aequorin Luminescence: Relation of Light Emission to Calcium Concentration—A Calcium-Independent Component

Allen, David G.; Blinks, John R.; Prendergast, Franklyn G.

doi:10.1126/science.841325

Cited by 353 publications

(233 citation statements)

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“…This algorithm was derived from a mathematical model proposed originally to explain from a molecular point of view the Ca 2+ -dependence of aequorin luminescence [20], but it can be used as a simple mathematical transformation independent of the model. The values for the parameters of the algorithm that should be used to calculate [Ca 2+ ] in experiments using ERmutAEQ reconstituted with coelenterazine n are: Regarding the significance of the parameters in the model [20], it is worth indicating that n was the number of Ca 2+ -binding sites in the model, and has a value of near 3 in native aequorin.…”

Section: Calibration Of Luminescence Datamentioning

confidence: 99%

“…The values for the parameters of the algorithm that should be used to calculate [Ca 2+ ] in experiments using ERmutAEQ reconstituted with coelenterazine n are: Regarding the significance of the parameters in the model [20], it is worth indicating that n was the number of Ca 2+ -binding sites in the model, and has a value of near 3 in native aequorin. The smaller value of n observed here is mainly due to the mutation of one of the Ca 2+ -binding sites, and corresponds with a decrease in the slope of the calibration curve (compare the slope of the AEQ1 curve with those of AEQ2 or AEQ3 ones in Fig.…”

Section: Calibration Of Luminescence Datamentioning

confidence: 99%

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Measuring [Ca2+] in the endoplasmic reticulum with aequorin

Álvarez

Montero

2002

Cell Calcium

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SummaryThe photoprotein aequorin was the first probe used to measure specifically the [Ca 2+ ] inside the lumen of the endoplasmic reticulum ([Ca 2+ ] ER ) of intact cells and it provides values for the steady-state [Ca 2+ ] ER , around 500 M, that closely match those obtained now by other procedures. Aequorin-based methods to measure [Ca 2+ ] ER offer several advantages: (i) targeting of the probe is extremely precise; (ii) the use of low Ca 2+ -affinity aequorin allows covering a large dynamic range of [Ca 2+ ], from 10 −5 to 10 −3 M; (iii) aequorin is nearly insensitive to changes in Mg 2+ or pH, has a high signal-to-noise ratio and calibration of the results in [Ca 2+ ] is made straightforward using a simple algorithm; and (iv) the equipment required for luminescence measurements in cell populations is simple and low-cost. On the negative side, this technique has also some disadvantages: (i) the relatively low amount of emitted light makes difficult performing single-cell imaging studies; (ii) reconstitution of aequorin with coelenterazine requires previous complete depletion of Ca 2+ of the ER for 1-2 h, a maneuver that may result in deleterious effects in some cells; (iii) because of the high rate of aequorin consumption at steady-state [Ca 2+ ] ER , only relatively brief experiments can be performed; and (iv) expression of ER-targeted aequorin requires previous transfection or infection to introduce the appropriate DNA construct, or alternatively the use of stable cell clones. Choosing aequorin or other techniques to measure [Ca 2+ ] ER will depend of the correct balance between these properties in a particular problem. © 2002 Elsevier Science Ltd. All rights reserved. HISTORICAL BACKGROUNDAlthough the ER has been known to be the main intracellular Ca 2+ store for nearly 20 years, monitoring the free [Ca 2+ ] in the ER ([Ca 2+ ] ER ) has proven to be a difficult task. The first studies using low-affinity fluorescent indicators required cell permeabilization to release the cytosolic dye and could not avoid compartmentalization of the dye into other organelles [1][2][3]. Apart of that, selectivity of these probes over Mg 2+ is poor and calibration was highly uncertain. On the other side, use of aequorin to measure [Ca 2+ ] ER has required solving two difficult technical problems: getting specific targeting of the probe into the ER and modifying the Ca 2+ -affinity of the probe to reach the adequate [Ca 2+ ] range.

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Section: Calibration Of Luminescence Datamentioning

confidence: 99%

Section: Calibration Of Luminescence Datamentioning

confidence: 99%

Measuring [Ca2+] in the endoplasmic reticulum with aequorin

Álvarez

Montero

2002

Cell Calcium

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“…where L is the luminescence intensity at any time point and L max is the integrated luminescence intensity (34).…”

Section: Methodsmentioning

confidence: 99%

An Osmotically Induced Cytosolic Ca2+ Transient Activates Calcineurin Signaling to Mediate Ion Homeostasis and Salt Tolerance of Saccharomyces cerevisiae

Matsumoto

Ellsmore

Cessna

et al. 2002

Journal of Biological Chemistry

139

119

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“…The residual aequorin was completely discharged by adding 200 mL of 20% ethanol containing 2 M CaCl 2 (final concentration 10% and 1 M, respectively). The resulting luminescence was used to estimate the total amount of aequorin present in various experiments to determine the rate of aequorin consumption for the calculation of the cytosolic Ca 21 concentrations according to Allen et al (1977).…”

Section: Aequorin-dependent Luminescence Measurementsmentioning

confidence: 99%

Effects of Feeding Spodoptera littoralis on Lima Bean Leaves. III. Membrane Depolarization and Involvement of Hydrogen Peroxide

et al. 2006

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In response to herbivore (Spodoptera littoralis) attack, lima bean (Phaseolus lunatus) leaves produced hydrogen peroxide (H 2 O 2 ) in concentrations that were higher when compared to mechanically damaged (MD) leaves. Cellular and subcellular localization analyses revealed that H 2 O 2 was mainly localized in MD and herbivore-wounded (HW) zones and spread throughout the veins and tissues. Preferentially, H 2 O 2 was found in cell walls of spongy and mesophyll cells facing intercellular spaces, even though confocal laser scanning microscopy analyses also revealed the presence of H 2 O 2 in mitochondria/peroxisomes. Increased gene and enzyme activations of superoxide dismutase after HW were in agreement with confocal laser scanning microscopy data. After MD, additional application of H 2 O 2 prompted a transient transmembrane potential (V m ) depolarization, with a V m depolarization rate that was higher when compared to HW leaves. In transgenic soybean (Glycine max) suspension cells expressing the Ca 21 -sensing aequorin system, increasing amounts of added H 2 O 2 correlated with a higher cytosolic calcium ([Ca 21 ] cyt ) concentration. In MD and HW leaves, H 2 O 2 also triggered the increase of [Ca 21 ] cyt , but MD-elicited [Ca 21 ] cyt increase was more pronounced when compared to HW leaves after addition of exogenous H 2 O 2 . The results clearly indicate that V m depolarization caused by HW makes the membrane potential more positive and reduces the ability of lima bean leaves to react to signaling molecules.

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Aequorin Luminescence: Relation of Light Emission to Calcium Concentration—A Calcium-Independent Component

Cited by 353 publications

References 13 publications

Measuring [Ca2+] in the endoplasmic reticulum with aequorin

Measuring [Ca2+] in the endoplasmic reticulum with aequorin

An Osmotically Induced Cytosolic Ca2+ Transient Activates Calcineurin Signaling to Mediate Ion Homeostasis and Salt Tolerance of Saccharomyces cerevisiae

Effects of Feeding Spodoptera littoralis on Lima Bean Leaves. III. Membrane Depolarization and Involvement of Hydrogen Peroxide

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