Botulinum neurotoxin A and neurotoxin E cleavage products of synaptosome-associated protein of 25 kd exhibit distinct actions on pancreatic islet beta-cell Kv2.1 channel gating.
OBJECTIVE
Synaptosome-associated protein of 25 kd (SNAP-25) regulates pancreatic islet beta-cell-delayed rectifier K channels (Kv2.1) in addition to insulin exocytosis. Botulinum neurotoxin A (BoNT/A) and E (BoNT/E) cleavage and presumed deletion of SNAP-25 have been used to examine SNAP-25 function. We hypothesized that proteolytic products of SNAP-25 (206 amino acids) resulting from BoNT/A and BoNT/E cleavage, SNAP-25(1-197) and SNAP-25(1-180), have independent actions on beta-cell Kv gating.


METHODS
We examined by confocal microscopy and immunoblotting BoNT/A and BoNT/E cleavage of SNAP-25 to these N-terminal fragments, and the consequent effects of these BoNTs and SNAP-25 fragments on Kv currents in rat beta cells and MIN6 cells by patch clamp electrophysiology.


RESULTS
Confocal microscopy and immunoblotting showed that MIN6 cells transfected with BoNT/A or BoNT/E generated SNAP-25(1-197) and SNAP-25(1-180) fragments that were retained in the cytosol. Both BoNTs caused increased rate of channel activation and slowed channel inactivation, mimicked by these SNAP-25 fragments, but not full-length SNAP-25. These SNAP-25 fragments potentiated tetraethylammonium block of beta-cell Kv currents.


CONCLUSIONS
BoNT/A or BoNT/E treatment of beta cells generates N-terminal SNAP-25 fragments that are retained in beta cells to directly influence Kv channel gating in a manner distinct from full-length SNAP-25, contributing to overall actions of these BoNTs on insulin secretion.
He
Yan
Y
Department of Medicine, University of Toronto, Toronto, Ontario, Canada.
Elias
Chadwick L
CL
Huang
Ya-Chi
YC
Gao
Xiaodong
X
Leung
Yuk-Man
YM
Kang
Youhou
Y
Xie
Huanli
H
Chaddock
John A
JA
Tsushima
Robert G
RG
Gaisano
Herbert Y
HY
D000818:Animals; D015153:Blotting, Western; D001905:Botulinum Toxins / Q000378:metabolism*; D019274:Botulinum Toxins, Type A / Q000378:metabolism*; D002460:Cell Line; D005982:Glutathione Transferase / Q000235:genetics; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000737:chemistry* / Q000502:physiology; D015640:Ion Channel Gating / Q000187:drug effects; D018613:Microscopy, Confocal; D018408:Patch-Clamp Techniques; D010446:Peptide Fragments / Q000235:genetics / Q000494:pharmacology*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics; D051669:Shab Potassium Channels / Q000187:drug effects* / Q000502:physiology; D050825:Synaptosomal-Associated Protein 25 / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D014162:Transfection
D007328:Insulin; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D051669:Shab Potassium Channels; D050825:Synaptosomal-Associated Protein 25; D005982:Glutathione Transferase; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; C096326:botulinum toxin type E