Journal
CELL CALCIUM
Volume 54, Issue 1, Pages 37-45Publisher
ELSEVIER SCI LTD
DOI: 10.1016/j.ceca.2013.04.001
Keywords
Aequorin; Endoplasmic reticulum; Ca2+; Ca2+-affinity; Ca2+-dynamics
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Funding
- Ministerio de Ciencia e Innovacion [BFU 2011-25763]
- Junta de Castilla y Leon [VA029A12-1]
- Spanish Ministerio de Ciencia e Innovacion
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We use here a new very low-Ca2+-affinity targeted aequorin to measure the [Ca2+] in the endoplasmic reticulum ([Ca2+](ER)). The new aequorin chimera has the right Ca2+-affinity to make long-lasting measurements of [Ca2+](ER) in the millimolar range. Moreover, previous Ca2+-depletion of the ER is no longer required. The steady-state [Ca2+](ER) obtained is 1-2 mM, higher than previously reported. In addition, we find evidence that there is significant heterogeneity in [Ca2+](ER) among different regions of the ER. About half of the ER had a [Ca2+](ER) of 1 mM or below, and the rest had [Ca2+](ER) values above 1 mM and in some parts even above 2 mM. About 5% of the ER was also found to have high [Ca2+](ER) levels but to be thapsigargin-insensitive and inositol trisphosphate insensitive. The rate of refilling with Ca2+ of the ER was almost linearly dependent on the extracellular [Ca2+] 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 [Ca2+] microdomains connecting the store operated Ca2+ channels with the ER Ca2+ pumps may be important during refilling. (C) 2013 Elsevier Ltd. All rights reserved.
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