Ca(2+) influx and cAMP elevation overcame botulinum toxin A but not tetanus toxin inhibition of insulin exocytosis

Previous reports showed that cleavage of vesicle-associatedmembrane protein-2 (VAMP-2) and synaptosomal-associated protein of 25 kDa (SNAP-25) by clostridial neurotoxins in permeabilized insulin-secreting -cells inhibited Ca²⁺-evoked insulinsecretion. In these reports, the solubleN-ethylmaleimide-sensitive factor attachment protein targetreceptor proteins might have formed complexes, which preclude fullaccessibility of the putative sites for neurotoxin cleavage. In thiswork, VAMP-2 and SNAP-25 were effectively cleaved before they formedtoxin-insensitive complexes by transient transfection of insulinoma HITor INS-1 cells with tetanus toxin (TeTx) or botulinum neurotoxin A(BoNT/A), as shown by immunoblotting and immunofluorescence microscopy. This resulted in an inhibition of Ca²⁺ (glucose orKCl)-evoked insulin release proportionate to the transfectionefficiency (40-50%) and an accumulation of insulin granules. Withthe use of patch-clamp capacitance measurements, Ca²⁺-evoked exocytosis by membrane depolarization to 10mV was abolished by TeTx (6% of control) but only moderately inhibitedby BoNT/A (30% of control). Depolarization to 0 mV to maximizeCa²⁺ influx partially overcame BoNT/A (50% of control) butnot TeTx inhibition. Of note, cAMP activation potentiatedCa²⁺-evoked secretion by 129% in control cells but only55% in BoNT/A-transfected cells and had negligible effects inTeTx-transfected cells. These results indicate that, whereas VAMP-2 isabsolutely necessary for insulin exocytosis, the effects of SNAP-25depletion on exocytosis, perhaps on insulin granule pool priming ormobilization steps, could be partially reversed by higher levels ofCa²⁺ or cAMP potentiation.