Munc-18 associates with syntaxin and serves as a negative regulator of exocytosis in the pancreatic beta -cell.
The Munc-18 protein (mammalian homologue of the unc-18 gene; also called nSec1 or rbSec1) has been identified as an essential component of the synaptic vesicle fusion protein complex. The cellular and subcellular localization and functional role of Munc-18 protein in pancreatic beta-cells was investigated. Subcellular fractionation of insulin-secreting HIT-T15 cells revealed a 67-kDa protein in both cytosol and membrane fractions. Immunohistochemistry showed punctate Munc-18 immunoreactivity in the cytoplasm of rat pancreatic islet cells. Direct double-labeling immunofluorescence histochemistry combined with confocal laser microscopy revealed the presence of Munc-18 immunoreactivity in insulin-, glucagon-, pancreatic polypeptide-, and somatostatin-containing cells. Syntaxin 1 immunoreactivity was detected in extracts of HIT-T15 cells, which were immunoprecipitated using Munc-18 antiserum, suggesting an intimate association of Munc-18 with syntaxin 1. Administration of Munc-18 peptide or Munc-18 antiserum to streptolysin O-permeabilized HIT-T15 cells resulted in significantly increased insulin release, but did not have any significant effect on voltage-gated Ca(2+) channel activity. The findings taken together show that the Munc-18 protein is present in insulin-secreting beta-cells and implicate Munc-18 as a negative regulator of the insulin secretory machinery via a mechanism that does not involve syntaxin-associated Ca(2+) channels.
Zhang
W
W
Department of Neuroscience, The Rolf Luft's Center for Diabetes Research, Karolinska Institutet, SE-171 77 Stockholm, Sweden.
Efanov
A
A
Yang
S N
SN
Fried
G
G
Kolare
S
S
Brown
H
H
Zaitsev
S
S
Berggren
P O
PO
Meister
B
B
D000818:Animals; D015153:Blotting, Western; D015220:Calcium Channels / Q000502:physiology; D005089:Exocytosis; D007150:Immunohistochemistry; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000502:physiology*; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins / Q000502:physiology*; D011233:Precipitin Tests; D050765:Qa-SNARE Proteins; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins
D015220:Calcium Channels; D007328:Insulin; D008565:Membrane Proteins; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D050765:Qa-SNARE Proteins; C494213:Stx1a protein, rat; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins