Functional measurements of [Ca2+] in the endoplasmic reticulum using a herpes virus to deliver targeted aequorin

Alonso, Marı́a Teresa; Barrero, María J.; Carnicero, Estela; Montero, Mayte; García‐Sancho, Javier; Alvarez, Javier

doi:10.1016/s0143-4160(98)90076-8

Cited by 73 publications

(61 citation statements)

References 37 publications

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“…Original measurements of [Ca 2+ ] ER had been made with mutated aequorin reconstituted with synthetic coelenterazine n. The values obtained in these original works for the steady-state [Ca 2+ ] ER in several cell types were between 300 and 800 M [1][2][3]18]. Later works by other authors reported similar values [4][5][6][7][8].…”

Section: Discussionmentioning

confidence: 91%

Ca2+ homeostasis in the endoplasmic reticulum measured with a new low-Ca2+-affinity targeted aequorin

et al. 2013

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a b s t r a c tWe use here a new very low-Ca 2+ -affinity targeted aequorin to measure the [Ca 2+ ] between 0.1 and 3 mM, and was only partially affected by mitochondrial membrane depolarization. Instead, it was significantly reduced by loading cells with chelators, and the fast chelator BAPTA was much more effective than the slow chelator EGTA. This suggests that local [Ca 2+ ] microdomains connecting the store operated Ca 2+ channels with the ER Ca 2+ pumps may be important during refilling.

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Section: Discussionmentioning

confidence: 91%

Ca2+ homeostasis in the endoplasmic reticulum measured with a new low-Ca2+-affinity targeted aequorin

et al. 2013

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“…The subsequent application of this technique to a wide range of cells was only possible by using viral methods to express the aequorin chimera [17]. Fig.…”

Section: The Problem Of the Ca 2+ -Affinitymentioning

confidence: 99%

“…1 shows the plasmid of herpes simplex virus type 1 used to clone ER-targeted aequorin and generate viruses able to express the construct. Thanks to this technique, it was possible to express ER-targeted aequorin in a variety of cell lines (NIH3T3, PC12, GH 3 ) and primary cultures (cerebellar granule cells, anterior pituitary cells and chromaffin cells), obtaining dynamic measurements of [Ca 2+ ] ER in these cells [17,18]. Fig.…”

Section: The Problem Of the Ca 2+ -Affinitymentioning

confidence: 99%

“…However, these methods are not efficient to express the construct in many other cell types, particularly primary cultures. To solve this problem, we have used defective herpes simplex virus (HSV1) carrying the gene, and this method has allowed to obtain a good expression in a series of cell lines and primary culture cells [17,18]. Adequate expression is usually obtained after 12 h using virus-based methods, and 18-24 h using transfection methods.…”

Section: Expression Of Er-targeted Mutated Aequorinmentioning

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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“…Taking into account the binding to cytosolic Ca 2+ buffers, we would expect a rate of 700/400 17.5 μmol · l cells −1 · s −1 , which is still much larger than the measured value, suggesting that more than 90% of the entering Ca 2+ is muffled [54] by transport out of the cytosol. Using aequorins targeted to different organelles delivered by a herpes virus [2], we were able to measure the contribution of the different transport processes [55]. Results are summarized in Fig.…”

Section: ]) the Ratio D[ T Ca]/d[camentioning

confidence: 99%

Mitochondria and chromaffin cell function

García‐Sancho

Diego

Garcı́a

2012

Pflugers Arch - Eur J Physiol

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Chromaffin cells are an excellent model for stimulus-secretion coupling. Ca 2+ entry through plasma membrane voltage-operated Ca 2+ channels (VOCC) is the trigger for secretion, but the intracellular organelles contribute subtle nuances to the Ca 2+ signal. The endoplasmic reticulum amplifies the cytosolic Ca 2+ ([Ca 2+ ] C ) signal by Ca 2+ -induced Ca 2+ release (CICR) and helps generation of microdomains with high [Ca 2+ ] C (HCMD) at the subplasmalemmal region. These HCMD induce exocytosis of the docked secretory vesicles. Mitochondria close to VOCC take up large amounts of Ca 2+ from HCMD and stop progression of the Ca 2+ wave towards the cell core. On the other hand, the increase of [Ca 2+ ] at the mitochondrial matrix stimulates respiration and tunes energy production to the increased needs of the exocytic activity. At the end of stimulation, [Ca 2+ ] C decreases rapidly and mitochondria release the Ca 2+ accumulated in the matrix through the Na + /Ca 2+ exchanger. VOCC, CICR sites and nearby mitochondria form functional triads that co-localize at the subplasmalemmal area, where secretory vesicles wait ready for exocytosis. These triads optimize stimulus-secretion coupling while avoiding propagation of the Ca 2+ signal to the cell core. Perturbation of their functioning in neurons may contribute to the genesis of excitotoxicity, ageing mental retardation and/or neurodegenerative disorders.

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Functional measurements of [Ca2+] in the endoplasmic reticulum using a herpes virus to deliver targeted aequorin

Abstract: SummaryChanges in the free calcium concentration of the endoplasmic reticulum ([Ca'+],,) play a central role controlling cellular functions like contraction, secretion or neuronal signaling. We recently reported that recombinant aequorin targeted to the endoplasmic reticulum (

Cited by 73 publications

References 37 publications

Ca2+ homeostasis in the endoplasmic reticulum measured with a new low-Ca2+-affinity targeted aequorin

Ca2+ homeostasis in the endoplasmic reticulum measured with a new low-Ca2+-affinity targeted aequorin

Measuring [Ca2+] in the endoplasmic reticulum with aequorin

Mitochondria and chromaffin cell function

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