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-associated membrane protein-2 (VAMP-2) and synaptosomal-associated protein of 25 kDa (SNAP-25) by clostridial neurotoxins in permeabilized insulin-secreting beta-cells inhibited Ca(2+)-evoked insulin secretion. In these reports, the soluble N-ethylmaleimide-sensitive factor attachment protein target receptor proteins might have formed complexes, which preclude full accessibility of the putative sites for neurotoxin cleavage. In this work, VAMP-2 and SNAP-25 were effectively cleaved before they formed toxin-insensitive complexes by transient transfection of insulinoma HIT or 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 Ca(2+) (glucose or KCl)-evoked insulin release proportionate to the transfection efficiency (40-50%) and an accumulation of insulin granules. With the use of patch-clamp capacitance measurements, Ca(2+)-evoked exocytosis by membrane depolarization to -10 mV was abolished by TeTx (6% of control) but only moderately inhibited by BoNT/A (30% of control). Depolarization to 0 mV to maximize Ca(2+) influx partially overcame BoNT/A (50% of control) but not TeTx inhibition. Of note, cAMP activation potentiated Ca(2+)-evoked secretion by 129% in control cells but only 55% in BoNT/A-transfected cells and had negligible effects in TeTx-transfected cells. These results indicate that, whereas VAMP-2 is absolutely necessary for insulin exocytosis, the effects of SNAP-25 depletion on exocytosis, perhaps on insulin granule pool priming or mobilization steps, could be partially reversed by higher levels of Ca(2+) or cAMP potentiation.
Huang
X
X
Department of Medicine, University of Toronto, Toronto M5S 1A8, Ontario, Canada M5G 1X8.
Kang
Y H
YH
Pasyk
E A
EA
Sheu
L
L
Wheeler
M B
MB
Trimble
W S
WS
Salapatek
A
A
Gaisano
H Y
HY
D000818:Animals; D019274:Botulinum Toxins, Type A / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002463:Cell Membrane Permeability; D002999:Clone Cells; D000242:Cyclic AMP / Q000378:metabolism*; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D005947:Glucose / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007340:Insulinoma; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology*; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000502:physiology*; D010190:Pancreatic Neoplasms; D018408:Patch-Clamp Techniques; D050683:R-SNARE Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25; D013744:Tetanus Toxin / Q000494:pharmacology*; D014162:Transfection; D014407:Tumor Cells, Cultured
D007328:Insulin; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050683:R-SNARE Proteins; D011994:Recombinant Proteins; D050825:Synaptosomal-Associated Protein 25; D013744:Tetanus Toxin; D000242:Cyclic AMP; D019274:Botulinum Toxins, Type A; D005947:Glucose; D002118:Calcium