Expression and localisation of synaptotagmin isoforms in endocrine β-cells: Their function in insulin exocytosis

Exocytosis of insulin containing Large Dense Core Vesicles (LDCVs) from pancreatic β-cells and derived cell lines is mainly controlled by Ca²⁺. Several lines of evidence have demonstrated a role of the Ca²⁺- and phospholipid-binding protein synaptotagmin (syt) in this event. Synaptotagmins form a large protein family with distinct affinities for Ca²⁺ determined by their two C₂ domains (C₂A/B). Except for the well-characterized isoforms I and II, their role is still unclear. We have used here insulin-secreting cells as a model system for LDCV exocytosis to gain insight into the function of synaptotagmins. Immunocytochemical analysis revealed that of the candidate Ca²⁺ sensors in LDCV exocytosis, syt III was not expressed in primary β-cells, whereas syt IV was only found adjacent to the TGN. However, syt V-VIII isoforms were expressed at different levels in various insulin-secreting cells and in pancreatic islet preparations. In streptolysin-O permeabilized primary β-cells the introduction of recombinant peptides (100 nM) corresponding to the C₂ domains of syt V, VII and VIII, but not of syt III, IV or VI, inhibited Ca²⁺-evoked insulin exocytosis by 30% without altering GTPγS-induced release. Our observations demonstrate that syt III and IV are not involved in the exocytosis of LDCVs from primary β-cells whereas V, VII and VIII may mediate Ca²⁺-regulation of exocytosis.