Scientific article

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

Published inBiochemical journal, vol. 339 ( Pt 1), p. 159-165
Publication date1999

The tSNARE (the target-membrane soluble NSF-attachment protein receptor, where NSF is N-ethylmaleimide-sensitive fusion protein) synaptosomal-associated protein of 25 kDa (SNAP-25) is expressed in pancreatic B-cells and its cleavage by botulinum neurotoxin E (BoNT/E) abolishes stimulated secretion of 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, using reverse transcriptase PCR, that messages for both SNAP-25 isoforms are expressed in primary pancreatic B and non-B cells as well as in insulin-secreting cell lines. After transfection, both isoforms can be detected at the plasma membrane as well as in an intracellular perinuclear region in the insulin-secreting cell line, HIT. To test for the functional role of the two isoforms in insulin secretion, mutant forms of SNAP-25a and b resistant against 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 insulin secretion can thus be investigated. To obtain the toxin-resistant mutant isoforms, the sequence around the BoNT/E cleavage site (R176QIDRIM182) was changed to P176QIKRIT182. This is the sequence of the equivalent region 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 toxin-treated HIT cells.

  • Animals
  • Base Sequence
  • Botulinum Toxins/pharmacology
  • Cell Line
  • DNA Primers
  • Humans
  • Insulin/ secretion
  • Islets of Langerhans/cytology/drug effects/ secretion
  • Membrane Proteins
  • Mutagenesis, Site-Directed
  • Nerve Tissue Proteins/genetics/ metabolism
  • Protein Isoforms/genetics/ metabolism
  • RNA, Messenger/genetics/metabolism
  • Rats
  • Synaptosomal-Associated Protein 25
Citation (ISO format)
GONELLE-GISPERT, C. et al. SNAP-25a and -25b isoforms are both expressed in insulin-secreting cells and can function in insulin secretion. In: Biochemical journal, 1999, vol. 339 ( Pt 1), p. 159–165. doi: 10.1042/bj3390159
Updates (1)
ISSN of the journal0264-6021

Technical informations

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