The Ca2+- and phospholipid-binding protein synaptotagmin is involved 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 μM cytosolic free Ca2+. The well-characterized synaptotagmin isoforms I and II are present in pancreatic β-cell lines RINm5F, INS-1 and HIT-T15 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 C2 domain of synaptotagmin I or II inhibited Ca2+-stimulated, but not GTPγS-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-T15 cells. However, mutations in the Ca2+-dependent phospholipid binding site of the first C2 domain (Δ180-183, D231S) again inhibited only Ca2+-, but not GTPγS-evoked exocytosis. In contrast, mutations in the IP4-binding sites of the second C2 domain (Δ325-341; K327,328,332Q) did not alter exocytosis. Synaptotagmin II mutated in both C2 domains (Δ180-183/K327,328,332Q) induced greater inhibition than mutant Δ180-183, suggesting a discrete requirement for the second C2 domain. Thus, synaptotagmin isoforms regulate exocytotic events occurring at low micromolar Ca2+.
- C domain
- Cytosolic Ca
- Insulin exocytosis
ASJC Scopus subject areas
- Molecular Biology
- Biochemistry, Genetics and Molecular Biology(all)
- Immunology and Microbiology(all)