Antibody inhibition of synaptosomal protein of 25 kDa (SNAP-25) and syntaxin 1 reduces rapid exocytosis in insulin-secreting cells.
SNARE-proteins (soluble NSF-attachment protein receptor) are important for Ca(2+)-dependent exocytosis. We have used capacitance measurements and confocal imaging to dissect the role of synaptosomal protein of 25 kDa (SNAP-25) and syntaxin 1 in rapid exocytosis in insulin-secreting pancreatic beta-cells. Following immunoneutralization of syntaxin 1 and SNAP-25, exocytosis was strongly reduced and associated with a marked reduction in the size of the readily releasable pool (RRP) by 65% and 86% in the presence of the anti-SNAP-25 and anti-syntaxin 1 antibodies respectively. The size of the immediately releasable pool (IRP), a subset of RRP in close association with the voltage-dependent Ca(2+)-channels, was reduced to an equal extent. The reduction in IRP correlated with slowed release kinetics and the time constant (tau) increased from a control value of 16 to 36 ms and 51 ms after inclusion of anti-SNAP-25 and anti-syntaxin 1 antibodies respectively in the pipette solution. We further show that SNAP-25 and syntaxin 1 aggregate in clusters along the plasma membrane. The size of these clusters was estimated to be approximately 300 nm and every beta-cell contained approximately 400 SNAP-25/syntaxin 1 clusters. Whereas the inhibitory action of the anti-syntaxin 1 antibody on exocytosis could be attributed almost entirely to suppression of the voltage-dependent Ca(2+)-current (-40%), the effect of the anti-SNAP-25 antibody was not mediated by decreased Ca(2+)-entry and is more likely due to a direct interference with the exocytotic machinery. Our data are consistent with the concept that both syntaxin 1 and SNAP-25 are required for rapid exocytosis in beta-cells.
Vikman
Jenny
J
Islet Cell Exocytosis, Department of Clinical Sciences in Malmo, Clinical Research Centre, Lund University, Ing. 72 UMAS, SE-20502 Malmö, Sweden.
Ma
Xiaosong
X
Hockerman
Gregory H
GH
Rorsman
Patrik
P
Eliasson
Lena
L
D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism; D002478:Cells, Cultured; D005089:Exocytosis / Q000502:physiology*; D007328:Insulin / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D018408:Patch-Clamp Techniques; D051381:Rats; D050825:Synaptosomal-Associated Protein 25 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D050827:Syntaxin 1 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*
D000906:Antibodies; D015220:Calcium Channels; D007328:Insulin; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D002118:Calcium