Please use this identifier to cite or link to this item: http://hdl.handle.net/1783.1/2264

Evaluation, using targeted aequorins, of the roles of the endoplasmic reticulum and its (Ca2++Mg2+)ATP-ases in the activation of store-operated Ca2+ channels in liver cells

Authors Chan, Chi Ming
Harland, M. Lyn
Webb, Sarah Elizabeth
Chen, Jinglong
Miller, Andrew Leitch
Barritt, Greg J.
Issue Date 2004
Source Cell calcium, v. 35, (4), 2004, APR, p. 317-331
Summary The process by which store-operated Ca2+ channels (SOCs) deliver Ca2+ to the endoplasmic reticulum (ER) and the role of (Ca2+ + Mg2+)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 Ca2+ in the ER ([Ca2+](er)) cells were pre-treated with 2,5-di-tert-butylhydroquinone (DBHQ) to deplete Ca2+ in the ER before reconstitution of holo-aequorin. The addition of extracellular Ca2+ (Ca-o(2+)) to Ca2+-depleted cells induced refilling of the ER, which was complete within 5 min. This was associated with a sharp transient increase in the cytoplasmic Ca2+ concentration ([Ca2+](cyt)) of about 15 s duration (a Ca-o(2+) -induced [Ca2+](cyt) spike) after which [Ca2+](cyt) remained elevated slightly above the basal value for a period of about 2 min (low [Ca2+](cyt) plateau). The Cao -induced [Ca2+](cyt) spike was inhibited by Gd3+, not affected by tetrakis-(2-pyridymethyl) ethylenediamine (TPEN), and broadened by ionomycin and the intracellular Ca2+ 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-o(2+) -induced [Ca2+](cyt) spike or the low [Ca2+](cyt) plateau whereas each inhibited the inflow of Ca2+ to the ER by about 80\%. Experiments conducted with carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) indicated that thapsigargin did not alter the amount of Ca2+ accumulated in mitochondria. The changes in [Ca2(+)]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 [Ca2+]cyt and (ii) thapsigargin and DBHQ inhibit the uptake of Ca2+ 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 another intracellular Ca2+ store), which contains thapsigargin-insensitive (Ca2+ + Mg2+)ATP-ases, in the activation of SOCs is briefly discussed. (C) 2003 Elsevier Ltd. All rights reserved.
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ISSN 0143-4160
Rights Cell calcium © copyright (2004) Elsevier. The Journal's web site is located at http://www.sciencedirect.com/
Language English
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