The first C-2 domain of synaptotagmin is required for exocytosis of insulin from pancreatic beta-cells: action of synaptotagmin at low micromolar calcium

The Ca2+- and phospholipid-binding protein synaptotagmin is invoked in neuroexocytosis. Its precise role and Ca2+-affinity in vivo are unclear, We investigated its putative function in insulin secretion which is maximally stimulated by 10 mu M cytosolic free Ca2+, The well-characterized synaptotagmin isoforms I and IT are present in pancreatic beta-cell lines RINm5F, INS-1 and HIT-TIS as shown by Northern and Western blots, Subcellular fractionation and confocal microscopy revealed their presence mainly on insulin-containing secretory granules whereas only minor amounts were found on synaptic vesicle-like microvesicles. Antibodies or Fab-fragments directed against the Ca2+-dependent phospholipid binding site of the first C-2 domain of synaptotagmin I or II inhibited Ca2+-stimulated, but not-GTP gamma S-induced exocytosis from streptolysin-O-permeabilized INS-1 and HIT-T15 cells, Transient expression of wild-type synaptotagmin II did not alter exocytosis in HIT-TIS cells. However, mutations in the Ca2+-dependent phospholipid binding site of the first C-2 domain (Delta 180-183, D231S) again inhibited only Ca2+-, but not GTP gamma S-evoked exocytosis. In contrast, mutations in the IP4-binding sites of the second C-2 domain (Delta 325-341; K327,328,332Q) did not alter exocytosis, Synaptotagmin Pi mutated in both C-2 domains (Delta 180-183/K327,328,332Q) induced greater inhibition than mutant Delta 180-183, suggesting a discrete requirement for the second C-2 domain, Thus, synaptotagmin isoforms regulate exocytotic events occurring at low micromolar Ca2+.