Ca2+ homeostasis in the endoplasmic reticulum measured with a new low-Ca2+-affinity targeted aequorin (original) (raw)
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Measuring [Ca2+] in the endoplasmic reticulum with aequorin
Cell Calcium, 2002
The 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.
Cell Calcium, 2004
The process by which store-operated Ca 2+ channels (SOCs) deliver Ca 2+ to the endoplasmic reticulum (ER) and the role of (Ca 2+ + Mg 2+ )ATP-ases of the ER in the activation of SOCs in H4-IIE liver cells were investigated using cell lines stably transfected with apo-aequorin targeted to the cytoplasmic space or the ER. In order to measure the concentration of Ca 2+ in the ER ([Ca 2+ ] er ), cells were pre-treated with 2,5di-tert-butylhydroquinone (DBHQ) to deplete Ca 2+ in the ER before reconstitution of holo-aequorin. The addition of extracellular Ca 2+ (Ca 2+ o ) to Ca 2+ -depleted cells induced refilling of the ER, which was complete within 5 min. This was associated with a sharp transient increase in the cytoplasmic Ca 2+ concentration ([Ca 2+ ] cyt ) of about 15 s duration (a Ca 2+ o -induced [Ca 2+ ] cyt spike) after which [Ca 2+ ] cyt remained elevated slightly above the basal value for a period of about 2 min (low [Ca 2+ ] cyt plateau). The Ca 2+ o -induced [Ca 2+ ] cyt spike was: inhibited by Gd 3+ , not affected by tetrakis-(2-pyridymethyl) ethylenediamine (TPEN), and broadened by ionomycin and the intracellular Ca 2+ chelators BAPTA and EGTA. Refilling of the ER was inhibited by caffeine. Neither thapsigargin nor DBHQ caused a detectable inhibition or change in shape of the Ca 2+ o -induced [Ca 2+ ] cyt spike or the low [Ca 2+ ] cyt plateau whereas each inhibited the inflow of Ca 2+ to the ER by about 80%. Experiments conducted with carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) indicated that thapsigargin did not alter the amount of Ca 2+ accumulated in mitochondria. The changes in [Ca 2+ ] cyt reported by aequorin were compared with those reported by fura-2. It is concluded that (i) there are significant quantitative differences between the manner in which aequorin and fura-2 sense changes in [Ca 2+ ] cyt and (ii) thapsigargin and DBHQ inhibit the uptake of Ca 2+ to the bulk of the ER but this is not associated with inhibition of the activation of SOCs. The possible involvement of a small sub-region of the ER (or -3another intracellular Ca 2+ store), which contains thapsigargin-insensitive (Ca 2+ + Mg 2+ )ATP-ases, in the activation of SOCs is briefly discussed. Abbreviations: ER, endoplasmic reticulum; SERCA, sarcoplasmic reticulum (Ca 2+ + Mg 2+ )ATP-ase; cytAEQ, aequorin targeted to the cytoplasmic space; erAEQ, aequorin targeted to the ER; [Ca 2+ ] cyt , the concentration of free Ca 2+ in the cytoplasmic space; [Ca 2+ ] er , the concentration of free Ca 2+ in the ER; Ca 2+ o , extracellular Ca 2+ ; KRB, Krebs-Ringer bicarbonate buffer; DBHQ, 2,5-di-tert-butylhydroquinone; BAPTA, 1,2bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; CCCP, carbonyl cyanide mchlorophenyl-hydrazone; PBS, phosphate buffered saline; HA1, haemaglutinin; TPEN, tetrakis-(2-pyridymethyl) ethylenediamine.
Two recombinant aequorin isoforms with different Ca 2 1 affinities, specifically targeted to the en- doplasmic reticulum (ER), were used in parallel to in- vestigate free Ca 2 1 homeostasis in the lumen of this or- ganelle. Here we show that, although identically and homogeneously distributed in the ER system, as re- vealed by both immunocytochemical and functional ev- idence, the two aequorins measured apparently very different concentrations of divalent cations ((Ca 2 1 ) er or (Sr 2 1 ) er ). Our data demonstrate that this contradiction is due to the heterogeneity of the (Ca 2 1 ) of the ae- quorin-enclosing endomembrane system. Because of the characteristics of the calibration procedure used to convert aequorin luminescence into Ca 2 1 concentra- tion, the (Ca 2 1 ) er values obtained at steady state tend, in fact, to reflect not the average ER values, but those of one or more subcompartments with lower (Ca 2 1 ). These subcompartments are not generated artefactu- ally during th...
The Journal of Cell Biology, 1997
Two recombinant aequorin isoforms with different Ca 2 ϩ affinities, specifically targeted to the endoplasmic reticulum (ER), were used in parallel to investigate free Ca 2 ϩ homeostasis in the lumen of this organelle. Here we show that, although identically and homogeneously distributed in the ER system, as revealed by both immunocytochemical and functional evidence, the two aequorins measured apparently very different concentrations of divalent cations ([Ca 2 ϩ ] er or [Sr 2 ϩ ] er ). Our data demonstrate that this contradiction is due to the heterogeneity of the [Ca 2 ϩ ] of the aequorin-enclosing endomembrane system. Because of the characteristics of the calibration procedure used to convert aequorin luminescence into Ca 2 ϩ concentration, the [Ca 2 ϩ ] er values obtained at steady state tend, in fact, to reflect not the average ER values, but those of one or more subcompartments with lower [Ca 2 ϩ ]. These subcompartments are not generated artefactually during the experiments, as revealed by the dynamic analysis of the ER structure in living cells carried out by means of an ER-targeted green fluorescent protein.
Cell Calcium, 1998
Changes 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 (ER) [Montero M., Brini M., Marsault R. et al. Monitoring dynamic changes in free Ca2+ concentration in the endoplasmic reticulum of intact cells. EMBO J 1995; 14: 5467-5475, Montero M., Barrero M.J., Alvarez J. [Ca2+] microdomains control agonist-induced Ca2+ release in intact cells. FASEB J 1997; 11: 881-8861 can be used to monitor selectively [Ca2+le, in intact HeLa cells. Here we have used a herpes simplex virus type 1 (HSV-1) based system to deliver targeted aequorin into a number of different cell types including both postmitotic primary cells (anterior pituitary cells, chromaffin cells and cerebellar neurons) and cell lines (HeLa, NIH3T3, GH, and PC12 cells). Functional studies showed that the steady state lumenal [Ca*+],, ranged from around 300 pM in granule cells to 800 ).rM in GH,cells. InsP,-coupled receptor stimulation with agonists like histamine (in HeLa, NIH3T3 and chromaffin cells), UTP and bradykinin (in PC12 cells) or thyrotropin-releasing hormone (TRH, in GH,cells) produced a very rapid decrease in lumenal [Ca'+],,. Caffeine caused a rapid Ca2+ depletion of the ER in chromaffin cells, but not in the other cell types. Depolarization by high K+ produced an immediate and reversible increase of [Ca2+lerin all the excitable cells (anterior pituitary, GH,, chromaffin cells and granule neurons)
Monitoring dynamic changes in free Ca2+ concentration in the endoplasmic reticulum of intact cells
The EMBO journal, 1995
Direct monitoring of the free Ca2+ concentration in the lumen of the endoplasmic reticulum (ER) is an important but still unsolved experimental problem. We have shown that a Ca(2+)-sensitive photoprotein, aequorin, can be addressed to defined subcellular compartments by adding the appropriate targeting sequences. By engineering a new aequorin chimera with reduced Ca2+ affinity, retained in the ER lumen via interaction of its N-terminus with the endogenous resident protein BiP, we show here that, after emptying the ER, Ca2+ is rapidly re-accumulated up to concentrations of > 100 microM, thus consuming most of the reporter photoprotein. An estimate of the steady-state Ca2+ concentration was obtained using Sr2+, a well-known Ca2+ surrogate which elicits a significantly slower rate of aequorin consumption. Under conditions in which the rate and extent of Sr2+ accumulation in the ER closely mimick those of Ca2+, the steady-state mean lumenal Sr2+ concentration ([Sr2+]er) was approxima...
Microscopy Research and Technique, 1999
In the last decade, the study of Ca 2ϩ homeostasis within organelles in living cells has been greatly enhanced by the utilisation of a recombinant Ca 2ϩ -sensitive photoprotein, aequorin. Aequorin is a Ca 2ϩ sensitive photoprotein of a coelenterate that, in the past, was widely employed to measure Ca 2ϩ concentration in living cells. In fact, the purified protein was widely used to monitor cytoplasmic [Ca 2ϩ ] changes in invertebrate muscle cells after microinjection. However, due to the time-consuming and traumatic procedure of microinjection, the role of aequorin in the study of Ca 2ϩ homeostasis remained confined to a limited number of cells (giant cells) susceptible to microinjection. Thus, in most instances, it was replaced by the fluorescent indicators developed by Roger Tsien and coworkers. The cloning of aequorin cDNA Proc. Natl. Acad. Sci. U.S.A. 82:3154-3158] and the explosive development of molecular biology offered new possibilities in the use of aequorin, as microinjection has been replaced by the simpler technique of cDNA transfection. As a polypeptide, aequorin allows the endogenous production of the photoprotein in cell systems as diverse as bacteria, yeast, slime molds, plants, and mammalian cells. Moreover, it is possible to specifically localise it within the cell by including defined targeting signals in the amino acid sequence. Targeted recombinant aequorins represent to date the most specific means of monitoring [Ca 2ϩ ] in subcellular organelles. In this review, we will not discuss the procedure of aequorin microinjection and its use as purified protein but we will present the new advances provided by recombinant aequorin in the study of intracellular Ca 2ϩ homeostasis, discussing in greater detail the advantages and disadvantages in the use of this probe.
Cell Calcium, 2007
In this study the relationship between the efficiency of endoplasmic reticulum (ER) Ca 2+ refilling and the extent of Ca 2+ entry was investigated in endothelial cells. ER and mitochondrial Ca 2+ concentration were measured using genetically encoded Ca 2+ sensors, while the amount of entering Ca 2+ was controlled by varying either the extracellular Ca 2+ or the electrical driving force for Ca 2+ by changing the plasma membrane potential. In the absence of an agonist, ER Ca 2+ replenishment was fully accomplished even if the Ca 2+ concentration applied was reduced from 2 to 0.5 mM. A similar strong efficiency of ER Ca 2+ refilling was obtained under condition of plasma membrane depolarization. However, in the presence of histamine, ER Ca 2+ refilling depended on mitochondrial Ca 2+ transport and was more susceptible to membrane depolarization. Store-operated Ca 2+ entry (SOCE), was strongly reduced under low Ca 2+ and depolarizing conditions but increased if ER Ca 2+ uptake was blocked or if ER Ca 2+ was released continuously by IP 3. A correlation of the kinetics of ER Ca 2+ refilling with cytosolic Ca 2+ signals revealed that termination of SOCE is a rapid event that is not delayed compared to ER refilling. Our data indicate that ER refilling occurs in priority to, and independently from the cytosolic Ca 2+ elevation upon Ca 2+ entry and that this important process is widely achieved even under conditions of diminished Ca 2+ entry.
Subcellular Ca2+ Dynamics Measured with Targeted Aequorin in Chromaffin Cells
Annals of the New York Academy of Sciences, 2002
In the last years, intracellular organella have emerged as key components in the generation and transduction of Ca2+ signals in adrenal chromaffin cells. Therefore, accurate measurements of Ca2+ inside cytoplasmic organella are essential for a comprehensive analysis of the Ca2+ redistribution that follows cell stimulation. We have en ineered the Cat+-sensitive photoprotein aequorin to monitor selectively C J + within the endoplasmic reticulum and the mitochondria. The targeted aequorins were delivered to the appropriate organelles of bovine chromaffin cells by using a herpes simplex virus-based amplicon vector, which permits efficient gene transfer and high levels of expression in infected cells. We have investigated the relationship between the caffeine and InsP3-sensitive Ca2+ pools and the presence of the Ca2+-induced Ca2+ release (CICR) mechanism in chromaffin cells. We find that ER Ca2+ pools responding to caffeine and to InsPJ mostly overlap and that CICR can be induced by Ca2+ entry elicited by high K+ depolarization. Moreover, the activation of Ca2+ channels, either the voltage-gated Cat+ channels on the plasma membrane or the channels on the endoplasmic reticulum (ER), generates subplasmalemmal high [Ca2+], domains that induce CaZ+ uptake by mitochondria. Interestingly, only a subpopulation of mitochondria, the one contained in the pool located close to the plasma membrane and the ryanodine receptors, take up Ca2+ efficiently, and the [Ca2+], reaches values of 300-500 pM.
The Biochemical journal, 1996
We describe a novel method to monitor the endoplasmic reticulum (ER) free Ca2+ in intact cells. Continuous perfusion of HeLa cells, expressing ER-targeted apoaequorin, with coelenterazine allowed the apoprotein to act as a pseudo-luciferase capable of reporting free Ca2+ from 0.1-100 microM. In intact HeLa cells, addition of ionomycin increased apoaequorin-generated light by 91%, indicating that resting ER free Ca2+ was approx. 2 microM. Agonist stimulation decreased the ER apoaequorin signal and proportionally increased cytosolic free Ca2+ consistent with agonist-induced release of Ca2+ from the ER.