title: Interactions between proteins implicated in exocytosis and voltage-gated calcium channels.
abstract: Neurotransmitter release from synaptic vesicles is triggered by voltage-gated calcium influx through P/Q-type or N-type calcium channels. Purification of N-type channels from rat brain synaptosomes initially suggested molecular interactions between calcium channels and two key proteins implicated in exocytosis: synaptotagmin I and syntaxin 1. Co-immunoprecipitation experiments were consistent with the hypothesis that both N- and P/Q-type calcium channels, but not L-type channels, are associated with the 7S complex containing syntaxin 1, SNAP-25, VAMP and synaptotagmin I or II. Immunofluorescence confocal microscopy at the frog neuromuscular junction confirmed that calcium channels, syntaxin 1 and SNAP-25 are co-localized at active zones of the presynaptic plasma membrane where transmitter release occurs. Experiments with recombinant proteins were performed to map synaptic protein interaction sites on the alpha 1A subunit, which forms the pore of the P/Q-type calcium channel. In vitro-translated 35S-synaptotagmin I bound to a site located on the cytoplasmic loop linking homologous domains II and III of the alpha 1A subunit. This direct link would target synaptotagmin, a putative calcium sensor for exocytosis, to a microdomain of calcium influx close to the channel mouth. Cysteine string proteins (CSPs) contain a J-domain characteristic of molecular chaperones that cooperate with Hsp70. They are located on synaptic vesicles and thought to be involved in modulating the activity of presynaptic calcium channels. CSPs were found to bind to the same domain of the calcium channel as synaptotagmin, and also to associate with VAMP. CSPs may act as molecular chaperones in association with Hsp70 to direct assembly or dissociation of multiprotein complexes at the calcium channel.
authors.affiliation: INSERM Unité 464, Institut Jean Roche, Faculté de Médecine Secteur Nord, Marseille, France. seagar.m@jean-roche.univ-mrs.fr
authors.forename: M
authors.initials: M
authors.lastname: Seagar
authors.forename: C
authors.initials: C
authors.lastname: Lévêque
authors.forename: N
authors.initials: N
authors.lastname: Charvin
authors.forename: B
authors.initials: B
authors.lastname: Marquèze
authors.forename: N
authors.initials: N
authors.lastname: Martin-Moutot
authors.forename: J A
authors.initials: JA
authors.lastname: Boudier
authors.forename: J L
authors.initials: JL
authors.lastname: Boudier
authors.forename: Y
authors.initials: Y
authors.lastname: Shoji-Kasai
authors.forename: K
authors.initials: K
authors.lastname: Sato
authors.forename: M
authors.initials: M
authors.lastname: Takahashi
mesh_terms: D000818:Animals; D000954:Antigens, Surface / Q000502:physiology; D015220:Calcium Channels / Q000145:classification / Q000302:isolation & purification / Q000502:physiology*; D002135:Calcium-Binding Proteins; D005089:Exocytosis / Q000502:physiology*; D050956:HSP40 Heat-Shock Proteins; D066298:In Vitro Techniques; D008562:Membrane Glycoproteins / Q000502:physiology; D008565:Membrane Proteins / Q000502:physiology; D008959:Models, Neurological; D009419:Nerve Tissue Proteins / Q000502:physiology*; D018377:Neurotransmitter Agents / Q000378:metabolism; D017729:Presynaptic Terminals / Q000502:physiology; D051381:Rats; D013572:Synaptic Vesicles / Q000502:physiology; D050825:Synaptosomal-Associated Protein 25; D050863:Synaptotagmin I; D050857:Synaptotagmins; D050827:Syntaxin 1
chemical_list: D000954:Antigens, Surface; D015220:Calcium Channels; D002135:Calcium-Binding Proteins; D050956:HSP40 Heat-Shock Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D018377:Neurotransmitter Agents; C494210:Snap25 protein, rat; C494213:Stx1a protein, rat; D050825:Synaptosomal-Associated Protein 25; D050863:Synaptotagmin I; D050827:Syntaxin 1; C494312:Syt1 protein, rat; C088460:cysteine string protein; D050857:Synaptotagmins