A peptide that mimics the carboxy-terminal domain of SNAP-25 blocks Ca(2+)-dependent exocytosis in chromaffin cells.
SNAP-25, a synaptosomal associated membrane protein of 25 kDa, participates in the presynaptic process of vesicle-plasma membrane fusion that results in neurotransmitter release at central nervous system synapses. SNAP-25 occurs in neuroendocrine cells and, in analogy to its role in neurons, has been implicated in catecholamine secretion, yet the nature of the underlying mechanism remains obscure. Here we use an anti-SNAP-25 monoclonal antibody to show that SNAP-25 is localized at the cytosolic surface of the plasma membrane of chromaffin cells. This antibody inhibited the Ca(2+)-evoked catecholamine release from digitonin-permeabilized chromaffin cells in a time- and dose-dependent manner. Remarkably, a 20-mer synthetic peptide representing the sequence of the C-terminal domain of SNAP-25 blocked Ca(2+)-dependent catecholamine release with an IC50 = 20 microM. The inhibitory activity of the peptide was sequence-specific as evidenced by the inertness of a control peptide with the same amino acid composition but random order. The C-terminal segment of SNAP-25, therefore, plays a key role in regulating Ca(2+)-dependent exocytosis, presumably mediated via interactions with other protein components of the fusion complex.
Gutiérrez
L M
LM
Departamento de Neuroquímica, Universidad de Alicante, Spain.
Cànaves
J M
JM
Ferrer-Montiel
A V
AV
Reig
J A
JA
Montal
M
M
Viniegra
S
S
D000313:Adrenal Medulla / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D004072:Digitonin; D005089:Exocytosis / Q000187:drug effects*; D005455:Fluorescent Antibody Technique; D008561:Membrane Fusion / Q000187:drug effects; D008565:Membrane Proteins; D008858:Microscopy, Phase-Contrast; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000737:chemistry / Q000276:immunology / Q000502:physiology*; D009638:Norepinephrine / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology*; D050825:Synaptosomal-Associated Protein 25
D000911:Antibodies, Monoclonal; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D050825:Synaptosomal-Associated Protein 25; D004072:Digitonin; D002118:Calcium; D009638:Norepinephrine