The first C2 domain of synaptotagmin is required for exocytosis of insulin from pancreatic β-cells: action of synaptotagmin at low micromolar calcium

Abstract

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⁺.