Interactions between proteins implicated in exocytosis and voltage-gated calcium channels

Neurotransmitter release from synaptic vesicles is triggered by voltage-gat ed calcium influx through P/Q-type or N-type calcium channels. Purification of N-type channels from rat brain synaptosomes initially suggested molecul ar interactions between calcium channels and two key proteins implicated in exocytosis: synaptotagmin I and syntaxin 1. Co-immunoprecipitation experim ents 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 conta ining syntaxin 1, SNAP-25, VAMP and synaptotagmin I or II. Immunofluorescen ce confocal microscopy at the frog neuromuscular junction confirmed that ca lcium channels, syntaxin 1 and SNAP-25 are co-localized at active zones of the presynaptic plasma membrane where transmitter release occurs. Experimen ts with recombinant proteins were performed to map synaptic protein interac tion sites on the alpha(1)A subunit, which forms the pore of the P/Q-type c alcium channel. In vitro-translated S-35-synaptotagmin I bound to a site lo cated on the cytoplasmic loop linking homologous domains II and III of the a,A subunit. This direct link would target synaptotagmin, a putative calciu m sensor for exocytosis, to a microdomain of calcium influx close to the ch annel mouth. Cysteine string proteins (CSPs) contain a J-domain characteris tic 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. CSP s may act as molecular chaperones in association with Hsp70 to direct assem bly or dissociation of multiprotein complexes at the calcium channel.