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Whole-cell patch-clamp experiments were performed to examine the mechanism underlying the inability of intracellular Ins(1,4,5)P(3) to activate the Ca(2+) release-activated Ca(2+) current (I(CRAC)) in rat basophilic leukaemia (RBL)-1 cells under conditions of weak cytoplasmic Ca(2+) buffering. Dialysis with Ins(1,4,5)P(3) in weak Ca(2+) buffer did not activate any macroscopic I(CRAC) even after precautions had been taken to minimize the extent of Ca(2+) entry during the experiment. Following intracellular dialysis with Ins(1,4,5)P(3) for >150 s in weak buffer, external application of the sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase (SERCA) pump blocker thapsigargin activated I(CRAC), and the current developed much more quickly than when thapsigargin was applied in the absence of Ins(1,4,5)P(3). This indicates that the Ins(1,4,5)P(3) receptors had not inactivated much over this timecourse. When external Ca(2+) was replaced by Ba(2+), Ins(1,4,5)P(3) still failed to generate any detectable I(CRAC) even though Ba(2+) permeates CRAC channels and is not taken up into the intracellular Ca(2+) stores. In strong Ca(2+) buffer, I(CRAC) could be activated by muscarinic-receptor stimulation, provided protein kinase C (PKC) was blocked. In weak buffer, however, as with Ins(1,4,5)P(3), stimulation of these receptors with carbachol did not activate I(CRAC) even after inhibition of PKC. The inability of Ins(1,4,5)P(3) to activate macroscopic I(CRAC) in weak Ca(2+) buffer was not altered by inhibition of Ca(2+)-dependent phosphorylation/dephosphorylation reactions. Our results suggest that the inability of Ins(1,4,5)P(3) to activate I(CRAC) under conditions of weak intracellular Ca(2+) buffering is not due to strong inactivation of the Ins(1,4,5)P(3) receptors. Instead, a futile Ca(2+) cycle across the stores seems to be occurring and SERCA pumps resequester sufficient Ca(2+) to ensure that the threshold for activation of macroscopic I(CRAC) has not been exceeded.

Type

Journal article

Journal

Biochem J

Publication Date

01/02/2001

Volume

353

Pages

561 - 567

Keywords

Animals, Barium, Buffers, Calcium, Calcium Channels, Calcium-Transporting ATPases, Endoplasmic Reticulum, Inositol 1,4,5-Trisphosphate Receptors, Leukemia, Basophilic, Acute, Phosphorylation, Rats, Receptors, Cytoplasmic and Nuclear, Sarcoplasmic Reticulum, Strontium, Tumor Cells, Cultured