Sarcoplasmic/endoplasmic-reticulum-Ca2+ -ATPase-mediated Ca2+ reuptake, and not Ins(1,4,5)P-3 receptor inactivation, prevents the activation of macroscopic Ca2+ release-activated Ca2+ current in the presence of physiologicalCa2+ buffer in rat basophilic leukaemia-1 cells

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 Ca2 + release-activated Ca2+ current (I-CRAC) in rat basophilic leukaemia (RBL) -1 cells under conditions of weak cytoplasmic Ca2+ buffering. Dialysis with Ins(1,4,5)P-3 in weak Ca2+ buffer did not activate any macroscopic I-CRAC, even after precautions had been taken to minimize the extent of Ca2+ 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/endop lasmic-reticulum Ca2+-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 Ca2+ was replaced by Ba2+, Ins(1,4,5)P-3 still failed to generate any dete ctable I-CRAC even though Ba2+ permeates CRAC channels and is not taken up into the intra- cellular Ca2+ stores. In strong Ca2+ buffer, I-CRAC could b e activated by muscarinic-receptor stimulation, provided protein kinase C ( PKC) was blocked. In weak buffer, however, as with Ins(1,4,5)P-3, stimulati on of these receptors with carbachol did not activate I-CRAC even after inh ibition of PKC. The inability of Ins(1.4,5)P-3 to activate macroscopic I-CR AC in weak Ca2+ buffer was not altered by inhibition of Ca2+-dependent phos phorylation/dephosphorylation reactions. Our results suggest that the inabi lity of Ins(1,4,5)P-3 to activate I-CRAC. under conditions of weak intracei lular Ca2+ buffering is not due to strong inactivation of the Ins(1,4,5)P-3 receptors. Instead, a futile Ca2+ cycle across the stores seems to be occu rring and SERCA pumps resequester sufficient Ca2+ to ensure that the thresh old for activation of macroscopic I-CRAC has not been exceeded.