Recombinant expression of the plasma membrane Na(+)/Ca(2+) exchanger affects local and global Ca(2+) homeostasis in Chinese hamster ovary cells.
AuthorsBrini M, Manni S, Carafoli E
JournalJ Biol Chem
PubMed ID12145298
'The cardiac type Na(+)/Ca(2+) exchanger (NCX1) has been transiently expressed in Chinese hamster ovary cells, which do not contain an endogenous exchanger, together with aequorin chimeras that are targeted to different intracellular compartments to investigate intracellular Ca(2+) homeostasis. The expression of NCX decreased the endoplasmic reticulum Ca(2+) concentration, [Ca(2+)](er), in ... More
Secretagogues modulate the calcium concentration in the endoplasmic reticulum of insulin-secreting cells. Studies in aequorin-expressing intact and permeabilized ins-1 cells.
AuthorsMaechler P, Kennedy ED, Sebö E, Valeva A, Pozzan T, Wollheim CB
JournalJ Biol Chem
PubMed ID10212237
'The precise regulation of the Ca2+ concentration in the endoplasmic reticulum ([Ca2+]er) is important for protein processing and signal transduction. In the pancreatic beta-cell, dysregulation of [Ca2+]er may cause impaired insulin secretion. The Ca2+-sensitive photoprotein aequorin mutated to lower its Ca2+ affinity was stably expressed in the endoplasmic reticulum (ER) ... More
Mitochondrial [Ca(2+)] oscillations driven by local high [Ca(2+)] domains generated by spontaneous electric activity.
AuthorsVillalobos C, Núñez L, Chamero P, Alonso MT, García-Sancho J
JournalJ Biol Chem
PubMed ID11668185
'Mitochondria take up calcium during cell activation thus shaping Ca(2+) signaling and exocytosis. In turn, Ca(2+) uptake by mitochondria increases respiration and ATP synthesis. Targeted aequorins are excellent Ca(2+) probes for subcellular analysis, but single-cell imaging has proven difficult. Here we combine virus-based expression of targeted aequorins with photon-counting imaging ... More
Preparation and handling of aequorin solutions for the measurement of cellular Ca2+.
AuthorsShimomura O
JournalCell Calcium
PubMed ID1959127
Main characteristics of the various types of aequorin presently available for measuring cellular Ca2+, i.e. heterogeneous aequorin, isoaequorins, recombinant aequorin, fluorescein-labeled aequorin and semi-synthetic aequorins, are summarized. Basic techniques of preparing and handling the solutions of those aequorins for measuring Ca2+, including such techniques as concentrating aequorin solutions, freeze-drying, changing ... More
Light-emitting properties of recombinant semi-synthetic aequorins and recombinant fluorescein-conjugated aequorin for measuring cellular calcium.
AuthorsShimomura O, Musicki B, Kishi Y, Inouye S
JournalCell Calcium
PubMed ID8519061
15 kinds of recombinant semi-synthetic aequorins and a recombinant fluorescein-conjugated aequorin were prepared and their properties in Ca(2+)-triggered luminescence were studied. The semi-synthetic aequorins showed a wide range of Ca(2+)-sensitivity. The luminescence intensity of a high-sensitivity type (hcp-aequorin) was greater than 10(4)-times that of a low-sensitivity type (n-aequorin) at pCa ... More
Membrane permeability of coelenterazine analogues measured with fish eggs.
AuthorsShimomura O
JournalBiochem J
PubMed ID9291095
To determine the suitability of various coelenterazine analogues for the regeneration of aequorin in living cells, the membrane permeabilities of 11 analogues were measured using the eggs of the killifish Fundulus grandis by soaking the eggs in solutions containing the analogues. The results indicated that e-coelenterazine, which has an exceptionally ... More
Ca2+-induced Ca2+ release in chromaffin cells seen from inside the ER with targeted aequorin.
AuthorsAlonso MT, Barrero MJ, Michelena P, Carnicero E, Cuchillo I, García AG, García-Sancho J, Montero M, Alvarez J
JournalJ Cell Biol
PubMed ID9922451
The presence and physiological role of Ca2+-induced Ca2+ release (CICR) in nonmuscle excitable cells has been investigated only indirectly through measurements of cytosolic [Ca2+] ([Ca2+]c). Using targeted aequorin, we have directly monitored [Ca2+] changes inside the ER ([Ca2+]ER) in bovine adrenal chromaffin cells. Ca2+ entry induced by cell depolarization triggered ... More
Dynamics of [Ca2+] in the endoplasmic reticulum and cytoplasm of intact HeLa cells. A comparative study.
AuthorsBarrero MJ, Montero M, Alvarez J
JournalJ Biol Chem
PubMed ID9346910
We have measured the [Ca2+] in the endoplasmic reticulum ([Ca2+]er) of intact HeLa cells at both 22 degrees C and 37 degrees C using endoplamsic reticulum-targeted, low Ca2+ affinity aequorin reconstituted with coelenterazine n. Aequorin consumption was much slower at 22 degrees C, and this allowed performing a much longer ... More
Imaging reversal of multidrug resistance in living mice with bioluminescence: MDR1 P-glycoprotein transports coelenterazine.
AuthorsPichler A, Prior JL, Piwnica-Worms D
JournalProc Natl Acad Sci U S A
PubMed ID14755051
Coelenterazine is widely distributed among marine organisms, producing bioluminescence by calcium-insensitive oxidation mediated by Renilla luciferase (Rluc) and calcium-dependent oxidation mediated by the photoprotein aequorin. Despite its abundance in nature and wide use of both proteins as reporters of gene expression and signal transduction, little is known about mechanisms of ... More
Direct monitoring of the calcium concentration in the sarcoplasmic and endoplasmic reticulum of skeletal muscle myotubes.
AuthorsRobert V, De Giorgi F, Massimino ML, Cantini M, Pozzan T
JournalJ Biol Chem
PubMed ID9804801
Direct monitoring of the free Ca2+ concentration in the sarcoplasmic reticulum (SR) was carried out in rat skeletal myotubes transfected with a specifically targeted aequorin chimera (srAEQ). Myotubes were also transfected with a chimeric aequorin (erAEQ) that we have demonstrated previously is retained in the endoplasmic reticulum (ER). Immunolocalization analysis ... More
The coxsackievirus 2B protein increases efflux of ions from the endoplasmic reticulum and Golgi, thereby inhibiting protein trafficking through the Golgi.
Authorsde Jong AS, Visch HJ, de Mattia F, van Dommelen MM, Swarts HG, Luyten T, Callewaert G, Melchers WJ, Willems PH, van Kuppeveld FJ
JournalJ Biol Chem
PubMed ID16540472
Coxsackievirus infection leads to a rapid reduction of the filling state of the endoplasmic reticulum (ER) and Golgi Ca2+ stores. The coxsackievirus 2B protein, a small membrane protein that localizes to the Golgi and to a lesser extent to the ER, has been proposed to play an important role in ... More
Dense core secretory vesicles revealed as a dynamic Ca(2+) store in neuroendocrine cells with a vesicle-associated membrane protein aequorin chimaera.
AuthorsMitchell KJ, Pinton P, Varadi A, Tacchetti C, Ainscow EK, Pozzan T, Rizzuto R, Rutter GA
JournalJ Cell Biol
PubMed ID11571310
The role of dense core secretory vesicles in the control of cytosolic-free Ca(2+) concentrations ([Ca(2+)](c)) in neuronal and neuroendocrine cells is enigmatic. By constructing a vesicle-associated membrane protein 2-synaptobrevin.aequorin chimera, we show that in clonal pancreatic islet beta-cells: (a) increases in [Ca(2+)](c) cause a prompt increase in intravesicular-free Ca(2+) concentration ... More
Ryanodine receptor type I and nicotinic acid adenine dinucleotide phosphate receptors mediate Ca2+ release from insulin-containing vesicles in living pancreatic beta-cells (MIN6).
AuthorsMitchell KJ, Lai FA, Rutter GA
JournalJ Biol Chem
PubMed ID12538591
We have demonstrated recently (Mitchell, K. J., Pinton, P., Varadi, A., Tacchetti, C., Ainscow, E. K., Pozzan, T., Rizzuto, R., and Rutter, G. A. (2001) J. Cell Biol. 155, 41-51) that ryanodine receptors (RyR) are present on insulin-containing secretory vesicles. Here we show that pancreatic islets and derived beta-cell lines ... More