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7 páginas, 5 figuras.-- El pdf es la versión post-print del artículo., STIM1 and Orai1 are the main players in capacitative calcium entry (CCE). STIM1 senses [Ca2+] inside the endoplasmic reticulum (ER) and, when it decreases, opens Orai1, a store-operated calcium channel (SOC) in the plasma membrane that promotes Ca2+ entry and increases cytosolic Ca2+. The final destination of the entering Ca2+ is the ER, which refills very efficiently (capacitatively) with it. We propose here that SERCA is the third element of CCE, to which is tightly coupled to favour rapid Ca2+ pumping from the high Ca2+ microdomains, generated at the SOC's mouth, to the ER. We find that, on depletion of the intracellular Ca2+ stores, SERCA co-localizes with STIM1 at puncta. Adequate coupling of CCE and ER Ca2+ pumping requires correct proportions of STIM1, Orai1 and SERCA. Overexpression of Orai1 decreased modestly Ca2+ entry, but produced a dramatic fall of Ca2+ uptake into ER, which was rescued by STIM1 co-expression or by increasing external Ca2+. In permeabilized cells, Ca2+ uptake into the ER was indistinguishable in the Orai1-expressing and in the control cells. We propose that excess Orai1 uncouples SERCA from Ca2+ entry in the intact cell by disturbing the fine topology of Ca2+ pumping complexes within the ER-plasma membrane junctions., This work was supported by grants from the Spanish Ministerio de Ciencia e Innovación (MICINN, BFI2007-60157 and SAF2009- 03175-E) and the Instituto de Salud Carlos III. IMM was supported by a fellowship from the Spanish MICINN.

Endomembranes modify the progression of the cytosolic Ca2+ wave and contribute to generate Ca2+ microdomains, both in the cytosol and inside the own organella. The concentration of Ca2+ in the cytosol ([Ca2+]C), the mitochondria ([Ca2+]M) and the nucleus ([Ca2+]N) are similar at rest, but may become very different during cell activation. Mitochondria avidly take up Ca2+ from the high [Ca2+]C microdomains generated during cell activation near Ca2+ channels of the plasma membrane and/or the endomembranes and prevent propagation of the high Ca2+ signal to the bulk cytosol. This shaping of [Ca2+]C signaling is essential for independent regulation of compartmentalized cell functions. On the other hand, a high [Ca2+]M signal is generated selectively in the mitochondria close to the active areas, which tunes up respiration to the increased local needs. The progression of the [Ca2+]C signal to the nucleus may be dampened by mitochondria, the nuclear envelope or higher buffering power inside the nucleoplasm. On the other hand, selective [Ca2+]N signals could be generated by direct release of stored Ca2+ into the nucleoplasm. Ca2+ release could even be restricted to subnuclear domains. Putative Ca2+ stores include the nuclear envelope, their invaginations inside the nucleoplasm (nucleoplasmic reticulum) and nuclear microvesicles. Inositol trisphosphate, cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate have all been reported to produce release of Ca2+ into the nucleoplasm, but contribution of these mechanisms under physiological conditions is still uncertain. © 2006 Elsevier Ltd. All rights reserved., This work was funded by grants from the Ministerio de Educación y Ciencia (BFU2004-02765/BFI and BFU2005-05464) and from Junta de Castilla y León (VA016A05). Pablo Chamero holded a predoctoral fellowship from the Basque government.

Changes in the free calcium concentration of the endoplasmic reticulum ([Ca2+]er) 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: 881-886] can be used to monitor selectively [Ca2+]er 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, GH3 and PC12 cells). Functional studies showed that the steady state lumenal [Ca2+]er ranged from around 300 microM in granule cells to 800 microM in GH3 cells. InsP3-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 GH3 cells) produced a very rapid decrease in lumenal [Ca2+]er. 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+]er in all the excitable cells (anterior pituitary, GH3, chromaffin cells and granule neurons). We conclude that delivery of recombinant aequorin to the ER using HSV amplicon provides the first direct quantitative and dynamic measurements of [Ca2+]er in several primary non-dividing cells.

The stimulus-secretion coupling for insulin secretion by pancreatic β cells in response to high glucose involves synchronic cytosolic calcium oscillations driven by bursting electrical activity. Calcium inside organelles can regulate additional functions, but analysis of subcellular calcium signals, specially at the single cell level, has been hampered for technical constrains. Here we have monitored nuclear calcium oscillations by bioluminescence imaging of targeted aequorin in individual cells within intact islets of Langerhans as well as in the whole islet. We find that glucose generates a pattern of nuclear calcium oscillations resembling those found in the cytosol. Some cells showed synchronous nuclear calcium oscillations suggesting that the islet of Langerhans may also regulate the activation of Ca2+ -responsive nuclear processes, such as gene transcription, in a coordinated, synchronic manner. The nuclear Ca2+ oscillations are due to bursting electrical activity and activation of plasma membrane voltage-gated Ca2+ channels with little or no contribution of calcium release from the intracellular Ca2+ stores. Irregularities in consumption of aequorins suggests that depolarization may generate formation of steep Ca2+ gradients in both the cytosol and the nucleus, but further research is required to investigate the role of such high [Ca2+] microdomains. © 2005 Elsevier Ltd. All rights reserved., This work was funded by grants from Ministerio de Educación y Ciencia (BFI 2001-2073, BFI2002-01469 and BFU2004-02765/BFI), Fondo de Investigaciones Sanitarias (FIS 03/1231) and RCMN (C03/08). C.V., L.N. and I.Q. are fellows of the Ramón y Cajal program from Ministerio de Educación y Ciencia, Spain. P.C. holds a predoctoral fellowship form the Basque government.