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

Jochen Lang, Mitsunori Fukuda, Hui Zhang, Katsuhiko Mikoshiba, Claes B. Wollheim

Research output: Contribution to journalArticle

100 Citations (Scopus)

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+.

Original languageEnglish
Pages (from-to)5837-5846
Number of pages10
JournalEMBO Journal
Volume16
Issue number19
DOIs
Publication statusPublished - 1997

Keywords

  • C domain
  • Cytosolic Ca
  • GTP
  • Insulin exocytosis
  • Synaptotagmin

ASJC Scopus subject areas

  • Neuroscience(all)
  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

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