Whole-cell patch-clamp experiments were performed to examine the mechanism underlying the inability of intracellular Ins(1,4,5)P3 to activate the Ca2+ release-activated Ca2+ current (ICRAC) in rat basophilic leukaemia (RBL)-1 cells under conditions of weak cytoplasmic Ca2+ buffering. Dialysis with Ins(1,4,5)P3 in weak Ca2+ buffer did not activate any macroscopic ICRAC even after precautions had been taken to minimize the extent of Ca2+ entry during the experiment. Following intracellular dialysis with Ins(1,4,5)P3 for > 150s in weak buffer, external application of the sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase (SERCA) pump blocker thapsigargin activated ICRAC, and the current developed much more quickly than when thapsigargin was applied in the absence of Ins(1,4,5)P3. This indicates that the Ins(1,4,5)P3 receptors had not inactivated much over this timecourse. When external Ca2+ was replaced by Ba2+, Ins(1,4,5)P3 still failed to generate any detectable ICRAC even though Ba2+ permeates CRAC channels and is not taken up into the intracellular Ca2+ stores. In strong Ca2+ buffer, ICRAC could be activated by muscarinic-receptor stimulation, provided protein kinase C (PKC) was blocked. In weak buffer, however, as with Ins(1,4,5)P3, stimulation of these receptors with carbachol did not activate ICRAC even after inhibition of PKC. The inability of Ins(1,4,5)P3 to activate macroscopic ICRAC in weak Ca2+ buffer was not altered by inhibition of Ca2+-dependent phosphorylation/dephosphorylation reactions. Our results suggest that the inability of Ins(1,4,5)P3 to activate ICRAC under conditions of weak intracellular Ca2+ buffering is not due to strong inactivation of the Ins(1,4,5)P3 receptors. Instead, a futile Ca2+ cycle across the stores seems to be occurring and SERCA pumps resequester sufficient Ca2+ to ensure that the threshold for activation of macroscopic ICRAC has not been exceeded.
Sarcoplasmic/endoplasmic-reticulum-Ca2+-ATPase-mediated Ca2+ reuptake, and not Ins(1,4,5)P3 receptor inactivation, prevents the activation of macroscopic Ca2+ release-activated Ca2+ current in the presence of physiological Ca2+ buffer in rat basophilic leukaemia-1 cells
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Daniel BAKOWSKI, Anant B. PAREKH; Sarcoplasmic/endoplasmic-reticulum-Ca2+-ATPase-mediated Ca2+ reuptake, and not Ins(1,4,5)P3 receptor inactivation, prevents the activation of macroscopic Ca2+ release-activated Ca2+ current in the presence of physiological Ca2+ buffer in rat basophilic leukaemia-1 cells. Biochem J 1 February 2001; 353 (3): 561–567. doi: https://doi.org/10.1042/bj3530561
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