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Title

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

AuthorsFuente, Sergio de la; Fonteriz, Rosalba I. ; Montero, Mayte ; Álvarez, Javier
KeywordsAequorin
Ca2+-dynamics
Ca2+
Ca2+-affinity
Endoplasmic reticulum
Issue Date2013
PublisherElsevier
CitationCell Calcium 54(1): 37-45 (2013)
AbstractWe 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 1-2mM, 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 1mM or below, and the rest had [Ca2+]ER values above 1mM and in some parts even above 2mM. 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 3mM, 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. © 2013 Elsevier Ltd.
URIhttp://hdl.handle.net/10261/116755
DOI10.1016/j.ceca.2013.04.001
Identifiersdoi: 10.1016/j.ceca.2013.04.001
issn: 0143-4160
e-issn: 1532-1991
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