title: Calmodulin and lipid binding to synaptobrevin regulates calcium-dependent exocytosis.
abstract: Neurotransmitter release involves the assembly of a heterotrimeric SNARE complex composed of the vesicle protein synaptobrevin (VAMP 2) and two plasma membrane partners, syntaxin 1 and SNAP-25. Calcium influx is thought to control this process via Ca(2+)-binding proteins that associate with components of the SNARE complex. Ca(2+)/calmodulin or phospholipids bind in a mutually exclusive fashion to a C-terminal domain of VAMP (VAMP(77-90)), and residues involved were identified by plasmon resonance spectroscopy. Microinjection of wild-type VAMP(77-90), but not mutant peptides, inhibited catecholamine release from chromaffin cells monitored by carbon fibre amperometry. Pre-incubation of PC12 pheochromocytoma cells with the irreversible calmodulin antagonist ophiobolin A inhibited Ca(2+)-dependent human growth hormone release in a permeabilized cell assay. Treatment of permeabilized cells with tetanus toxin light chain (TeNT) also suppressed secretion. In the presence of TeNT, exocytosis was restored by transfection of TeNT-resistant (Q(76)V, F(77)W) VAMP, but additional targeted mutations in VAMP(77-90) abolished its ability to rescue release. The calmodulin- and phospholipid-binding domain of VAMP 2 is thus required for Ca(2+)-dependent exocytosis, possibly to regulate SNARE complex assembly.
authors.affiliation: Institut National de la Santé et de la Recherche Médicale Unité 464, Université de la Méditerranée, 13916 Marseille Cedex 20, France.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Quetglas
authors.forename: Cecile
authors.initials: C
authors.lastname: Iborra
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Sasakawa
authors.forename: Luc
authors.initials: L
authors.lastname: De Haro
authors.forename: Konosuke
authors.initials: K
authors.lastname: Kumakura
authors.forename: Kazuki
authors.initials: K
authors.lastname: Sato
authors.forename: Christian
authors.initials: C
authors.lastname: Leveque
authors.forename: Michael
authors.initials: M
authors.lastname: Seagar
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002147:Calmodulin / Q000737:chemistry / Q000502:physiology*; D002395:Catecholamines / Q000378:metabolism*; D002417:Cattle; D019439:Chromaffin Cells / Q000187:drug effects / Q000378:metabolism*; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D019382:Human Growth Hormone / Q000378:metabolism; D006801:Humans; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology / Q000502:physiology*; D008666:Metalloendopeptidases / Q000494:pharmacology; D008845:Microinjections; D008969:Molecular Sequence Data; D016716:PC12 Cells / Q000187:drug effects / Q000378:metabolism; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000494:pharmacology; D010743:Phospholipids / Q000502:physiology*; D017434:Protein Structure, Tertiary; D050683:R-SNARE Proteins; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050600:SNARE Proteins; D054830:Sesterterpenes; D020349:Surface Plasmon Resonance; D050827:Syntaxin 1; D013729:Terpenes / Q000494:pharmacology; D013744:Tetanus Toxin / Q000494:pharmacology; D014162:Transfection; D033921:Vesicular Transport Proteins
chemical_list: D002147:Calmodulin; D002395:Catecholamines; D046911:Macromolecular Substances; D008565:Membrane Proteins; D010446:Peptide Fragments; D010743:Phospholipids; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins; C494212:STX1A protein, human; D054830:Sesterterpenes; C494213:Stx1a protein, rat; D050827:Syntaxin 1; D013729:Terpenes; D013744:Tetanus Toxin; D033921:Vesicular Transport Proteins; D019382:Human Growth Hormone; C040764:ophiobolin A; D008666:Metalloendopeptidases; C093556:zinc-endopeptidase, tetanus neurotoxin