Metabolism and biological activities of lnsP5 and InsP6 in neuronal cells

Several kinds of stimulants are known to induce inositol 1，3，4，5，6-pentakisphosphate(InsP5)and inositol hexakisphosphate(InsP6)accumulations in intact adrenal chromaffin cells，N1E-115cells and rat cerebellar granule cells。Recently，we suggested that the binding of inositol polyphosphates to the C2B domain of synaptotagmin may clamp spontaneous fusion of the docked or primed vesicles in adrenal chromaffin cells.In this study，we investigated the Ca I D12+ii D1-evoked release of InsP5 and InsP6，and the effects of the antibodies against synaptotagmin C2A and C2B domains in digitonin-permeabilized adrenal chromaffin cells。When the cells were stimulated with Ca I D12+ii D1(100µM)，transient increase of InsP5 and InsP6 in the cytosolic medium were observed。Upon a Ca I D12+I D1-challenge，amount of InsP5 and InsP6 in the cytosolic medium reached the maximum at 15 sec.Anti-C2A antibody(C2A Ab)completely inhibited Ca I D12+ii D1-induced InsP5 and InsP6 accumulation in cytosolic medium。Anti-C2B antibody(C2B Ab)also inhibited Ca I D12+ii D1-induced InsP5 and InsP6 accumulation in the cytosolic medium。Since pretreatment with C2B Ab during permeabilization，but not with C2A Ab，induced increase of InsP5 and InsP6in the permeabilizing buffer，the mechanisms of the inhibitory effects of the C2B Ab is different from those of C2A Ab。These results strongly suggest that the binding of increased intracellular Ca I D12+I D 1 to the C2A domain liberate InsP5 and InsP6from the C2B domain of synaptotagmin，supporting our idea of the clamp of fusion by synaptotagmin and inositol polyphosphates.

Several kinds of stimulants are known to induce inositol1,3,4,5,6-pentakisphosphate (InsP5) and inositol hexakisphosphate (InsP6) accumulations in intact adrenal chromaffin cells, N1E-115 cells and rat cerebellar granule cells. Recently, we suggested that the binding of inositol polyphosphates to the C2B domain of synaptotagmin may clamp spontaneous fusion of the docked or primed vesicles in adrenal chromaffin cells. In this study, we investigated the CaィイD12+ィエD1-evoked release of InsP5 and InsP6, and the effects of the antibodies against synaptotagmin C2A and C2B domains in digitonin-permeabilized adrenal chromaffin cells. When the cells were stimulated with CaィイD12+ィエD1 (100μM), transient increase of InsP5 and InsP6 in the cytosolic medium were observed. Upon a CaィイD12+ィエD1-challenge, amount of InsP5 and InsP6 in the cytosolic medium reached the maximum at 15 sec. Anti-C2A antibody (C2A Ab) completely inhibited CaィイD12+ィエD1-induced InsP5 and InsP6 accumulation in cytosolic medium. Anti-C2B antibody (C2B Ab) also inhibited CaィイD12+ィエD1-induced InsP5 and InsP6 accumulation in the cytosolic medium. Since pretreatment with C2B Ab during permeabilization, but not with C2A Ab, induced increase of InsP5 and InsP6 in the permeabilizing buffer, the mechanisms of the inhibitory effects of the C2B Ab is different from those of C2A Ab. These results strongly suggest that the binding of increased intracellular CaィイD12+ィエD1 to the C2A domain liberate InsP5 and InsP6 from the C2B domain of synaptotagmin, supporting our idea of the clamp of fusion by synaptotagmin and inositol polyphosphates.