Identification of intracellular calcium pools - Selective modification by thapsigargin

Junhui Bian, Tarun K. Ghosh, Jian Cheng Wang, Donald Gill

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Abstract

Recent studies have identified inositol 1,4,5-trisphosphate(InsP3)-sensitive and -insensitive Ca2+ pools and a GTP-dependent mechanism that transfers Ca2+ between them. Here, the Ca2+ pump-inhibitory sesquiterpene lactone, thapsigargin, is shown to distinguish these two Ca2+ pools and identify a third Ca2+ pumping pool unresponsive to InsP3 or GTP. Using saponinpermeabilized DDT1 MF-2 smooth muscle cells, approximately 75% of total intracellular ATP-dependent Ca2+ accumulation is blocked by thapsigargin with an IC50 of 30 nM. In contrast, 1 mM vanadate or 5 μM A23 187 block 100% of Ca2+ accumulation. The thapsigargin-responsive Ca2+ pool corresponds exactly to that released by 10 μM InsP3 in the presence of 10 μM GTP. Indeed, addition of InsP3 with GTP has no effect on Ca2+ accumulated in the presence of 3 μM thapsigargin whereas A23187 releases all the remaining Ca2+. Added after maximal Ca2+ uptake, thapsigargin induces only slow Ca2+ release consistent with blockade of pumping activity. Unlike InsP3, the action of thapsigargin is entirely heparin insensitive. The large increment in Ca2+ uptake caused by 12 mM oxalate is completely reversed by thapsigargin, indicating that thapsigargin functions on an oxalate-permeable pool. Moreover, the still larger uptake induced by GTP in the presence of oxalate is also completely reversed by either thapsigargin or InsP3. The results indicate that thapsigargin blocks Ca2+ uptake into two discrete pools: the InsP3-sensitive, oxalate-permeable Ca2+ pool and the InsP3-insensitive, oxalate-impermeable Ca2+ pool that can be "recruited" into the InsP3-sensitive pool by GTP-dependent Ca2+ translocation (Ghosh, T. K., Mullaney, J. M., Tarazi, F. I., and Gill, D. L. (1989) Nature 340, 236-239). Additionally, a third Ca2+ pool is defined, unreleasable by InsP3 or GTP, and containing a thapsigargin-insensitive Ca2+ pump.

Original languageEnglish (US)
Pages (from-to)8801-8806
Number of pages6
JournalJournal of Biological Chemistry
Volume266
Issue number14
Publication statusPublished - 1991

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Bian, Junhui ; Ghosh, Tarun K. ; Wang, Jian Cheng ; Gill, Donald. / Identification of intracellular calcium pools - Selective modification by thapsigargin. In: Journal of Biological Chemistry. 1991 ; Vol. 266, No. 14. pp. 8801-8806.