SNAP-25a and -25b isoforms are both expressed in insulin-secreting cells and can function in insulin secretion

The tSNARE (the target-membrane soluble NSF-attachment protein receptor, wh ere NSF is N-ethylmaleimide-sensitive fusion protein) synaptosomal-associat ed protein of 25 kDa (SNAP-25) is expressed in pancreatic B-cells and its c leavage by botulinum neurotoxin E (BoNT/E) abolishes stimulated secretion o f insulin, In the nervous system, two SNAP-25 isoforms (a and b) have been described that are produced by alternative splicing. Here it is shown, usin g reverse transcriptase PCR, that messages for both SNAP-25 isoforms are ex pressed in primary pancreatic B and non-B cells as well as in insulin-secre ting cell lines. After transfection, both isoforms can be detected at the p lasma membrane as well as in an intracellular perinuclear region in the ins ulin-secreting cell line, HIT. To test for the functional role of the two i soforms in insulin secretion, mutant forms of SNAP-25a and b resistant agai nst cleavage by BoNT/E were generated. Such mutant SNAP-25, when expressed in HIT cells, is not inactivated by BoNT/E and its ability to restore insul in secretion can thus be investigated. To obtain the toxin-resistant mutant isoforms, the sequence around the BoNT/E cleavage site (R(176)QIDRIM(182)) was changed to P(176)QIKRIT(182). This is the sequence of the equivalent r egion of human SNAP-23 ((P187-T194)), which has been shown to be resistant to BoNT/E. The mutant SNAP-25 was resistant to BoNT/E in vitro and in vivo and both mutant isoforms were able to reconstitute insulin secretion from t oxin-treated HIT cells.