Synaptotagmins (Syts) serve as a Ca 2+ sensor in the release of neurotransmitters and hormones. Inositol polyphosphates (InsPPs) such as Inositol 1,3,4,5,6-pentakisphosphate (InsP 5) and inositol hexakisphosphate (InsP 6) bind to Ca 2+-binding C2B domain of Syt I and II, and inhibit transmitter release. We have shown that the inhibition by InsPPs is reversed by Ca 2+ in adrenal chromaffin cells, while a rapid accumulation of endogenous InsP 5 and InsP 6 upon depolarizing stimuli have been reported in these and some other cells. Such a rapid accumulation of InsPPs, if not all, might reflect their dissociation from C2B domain of Syt. To elucidate the functional relevance, we studied the effects of antibodies against C2A and C2B domains (anti-C2A Ab, anti-C2B Ab) on the accumulation of InsPPs induced by Ca 2+ in digitonin-permeabilized adrenal chromaffin cells. Anti-C2B Ab by itself caused an accumulation of InsPPs in the permeabilizing medium, and increased spontaneous release of catecholamines (CA). Anti-C2A Ab abolished Ca 2+-induced increase of InsPPs in cytosolic component, and inhibited Ca 2+-evoked release of CA with little effect on the spontaneous release. Microinjection of InsP 6 but not inositol hexakissulfate into intact chromaffin cells inhibited both spontaneous and nicotine-evoked exocytotic events. These results suggest that endogenous InsPPs bound to the C2B domain clamp spontaneous fusion of the docked or primed vesicles at resting level of intracellular Ca 2+, and binding of Ca 2+ to the C2A or/and C2B domain facilitate fusion dissociating InsPPs from Syt in adrenal chromaffin cells. This article is part of a Special Issue entitled 'Trends in Neuropharmacology: In Memory of Erminio Costa'.
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