TRUNCATED SNAP-25 (1-197), LIKE BOTULINUM-NEUROTOXIN-A, CAN INHIBIT INSULIN-SECRETION FROM HIT-T15 INSULINOMA CELLS

We and others have previously shown that insulin-secreting cells of th e pancreas express high levels of SNAP-25 (synaptosomal-associated pro tein of 25 kDa), a 206-amino acid t-SNARE (target soluble N-ethylmalei mide-sensitive factor attachment protein receptors) implicated in syna ptic vesicle exocytosis. In the present study, we show that SNAP-25 is required for insulin secretion by transient transfection of Botulinum Neurotoxin A (BoNT/A) into insulin-secreting HIT-T15 cells. Transient expression of BoNT/A cleaved the endogenous as well as overexpressed SNAP-25 proteins and caused significant reductions in K+ and glucose-e voked secretion of insulin. To determine whether the inhibition of rel ease was due to the depletion of functional SNAP-25 or the accumulatio n of proteolytic by-products, we transfected cells with SNAP-25 protei ns from which the C-terminal nine amino acids had been deleted to mimi c the effects of the toxin. This modified SNAP-25 (amino acids 1-197) remained bound to the plasma membrane but was as effective as the toxi n at inhibiting insulin secretion. Microfluorimetry revealed that the inhibition of secretion was due neither to changes in basal cytosolic Ca2+ levels nor in Ca2+ influx evoked by K+-mediated plasma membrane d epolarization. Electron microscopy revealed that cells transfected wit h either BoNT/A or truncated SNAP-25 contained significantly higher nu mbers of insulin granules, many of which clustered close to the plasma membrane. Together, these results demonstrate that functional SNAP-25 proteins are required for insulin secretion and suggest that the inhi bitory action of BoNT/A toxin on insulin secretion is in part caused b y the production of the plasma membrane-bound cleavage product, which itself interferes with insulin granule docking and fusion.