[Ca2+] microdomains control agonist-induced Ca2+ release in intact HeLa cells

We have monitored specifically the [Ca2+] in the lumen of the endoplasmic reticulum (ER) of intact HeLa cells using-an ER-targeted low Ca2+-affinity aequorin. The steady-state [Ca2+] in the ER was around 600 mu M Histamine induced a concentration-dependent decrease in lumenal [Ca2+], whose rate increased near one order of magnitude and became ''quantal'' when cytosolic [Ca2+] ([Ca2+](c)) was clamped with the Ca2+ chelator BAPTA. This effect was not due to decreased Ca2+ pumping because simultaneous addition of a SERCA inhibitor produced only additive effects, Given that inhibition by [Ca2+](c) of the inositol 1,4,5-trisphosphate-gated channels requires a [Ca2+](c) much higher than that observed in the bulk cytosol after histamine addition, we conclude that local [Ca2+](c) microdomains at the site of release strongly inhibit agonist-induced Ca2+ mobilization in intact cells, This effect should play a key role in the mechanism controlling cytosolic [Ca2+] oscillations and waves, and therefore in the generation of spatio-temporal Ca2+ patterns.