Denise Walker, Fré dé rique Berton, specifically expressed in distinct patterns in neural and Cé cile Raymond, Masakazu Kataoka 1 , neuroendocrine tissues (Ullrich et al., 1994;Marquèze et al., Yoko Shoji-Kasai 1 , Masami Takahashi 1 , Berton et al., 1997). Synaptotagmin I, a major mem- 1992, 1994 Yoshida et al., 1992;El Far et al., 1995) and to vesicle fusion and exocytosis of neurotransmitters. MichelP/Q-type (Martin-Moutot et al., 1996), but not L-type calThe interaction of synaptotagmins with native P/Q-type cium channels (El Far et al., 1995). Furthermore, co-exprescalcium channels was studied in solubilized synaptosion of syntaxin with calcium channels induces a somes from rat cerebellum. Antibodies against synaptomodification of current gating properties displaying a tagmins I and II, but not IV co-immunoprecipitated similar specificity for N-or P/Q-type channels [ 125 I]ω-conotoxin MVIIC-labelled calcium channels. (Bezprozvanny et al., 1995). These findings are consistent Direct interactions were studied between in vitro-transwith observations that neurotransmitter release at many lated [ 35 S]synaptotagmin I and fusion proteins concentral synapses is blocked by antagonists of N-or P/Qtaining cytoplasmic loops of the α 1 A subunit (BI type calcium channels, but is insensitive to inhibitors of isoform). Gel overlay revealed the association of L-type channels (Takahashi and Momiyama, 1993; Wheeler synaptotagmin I with a single region (residues 780-969) et al., 1994). Neuronal calcium channels are heteromeric located in the intracellular loop connecting homologous proteins constituted by an α 1 subunit which forms the transdomains II and III. Saturable calcium-independent membrane pore, associated with auxiliary α 2 δ and β subbinding occurred with equilibrium dissociation conunits (Birnbaumer et al., 1994). Association with core stants of 70 nM and 340 nM at 4°C and pH 7.4, and complexes involves syntaxin and SNAP25 binding to α 1 subunits on the cytoplasmic loop that links homologous association was blocked by addition of excess recombindomains II and III (Sheng et al., 1994(Sheng et al., , 1996; Rettig et al., ant synaptotagmin I. Direct synaptotagmin binding to 1996). the pore-forming subunit of the P/Q-type channel may It has been suggested that interactions between synaptic optimally locate the calcium-binding sites that initiate protein complexes and calcium channels may optimally exocytosis within a zone of voltage-gated calcium entry.locate the calcium sensor synaptotagmin within domains

P-and Q-type calcium channels, which trigger rapid neurotransmitter release at many mammalian synapses, are blocked by -conotoxin MVIIC. 125I--Conotoxin MVIIC binding to rat cerebellar synaptosomes was not displaced by -conotoxins GVIA or MVIIA (K i > 1 M), which are selective for N-type calcium channels. Solubilized 125 I--conotoxin MVIIC receptors were specifically recognized by antibodies directed against ␣ 1 A calcium channel subunits, proteins known to constitute a pore with P/Q-like channel properties. Antibodies against syntaxin 1, SNAP 25, and VAMP 2 (synaptobrevin) each immunoprecipitated a similar fraction (20 -40%) of -conotoxin MVIIC receptors. Immunoprecipitation was not additive, suggesting that heterotrimeric (SNARE) complexes containing these three proteins interact with P/Q-type calcium channels. Immobilized monoclonal anti-syntaxin antibodies retained ␣ 1 A calcium channel subunits of 220, 180 and 160 kDa monitored by immunoblotting with site directed antibodies. Synaptotagmin was detected in channel-associated complexes, but not synaptophysin, Rab 3A nor rat cysteine string protein. Trimeric SNARE complexes are implicated in calcium-dependent exocytosis, a process thought to be regulated by synaptotagmin. Our results indicate that these proteins interact with P/Q-type calcium channels, which may optimize their location within domains of calcium influx.Neuronal calcium channels are heteromeric proteins constituted by an ␣ 1 subunit, which forms the voltage-gated transmembrane pore, associated with auxiliary ␣ 2 ␦ and ␤ subunits. Five genes encoding homologous ␣ 1 subunits (␣ 1 A-E) with different channel properties are expressed in the rat brain (reviewed by Snutch and Reiner (1992) and Birnbaumer et al. (1994)). ␣ 1 C and ␣ 1 D subunits each form 1,4-dihydropyridinesensitive L-type channels, whereas ␣ 1 B subunits constitute N-type channels that are specifically blocked by -conotoxins GVIA or MVIIA (GVIA, MVIIA).

Neurotransmitter release from synaptic vesicles is triggered by voltage-gated calcium in£ux through P/Q-type or N-type calcium channels. Puri¢cation 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. Immuno£uorescence confocal microscopy at the frog neuromuscular junction con¢rmed 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 a 1 A subunit, which forms the pore of the P/Q-type calcium channel. In vitro-translated 35 S-synaptotagmin I bound to a site located on the cytoplasmic loop linking homologous domains II and III of the a 1 A subunit. This direct link would target synaptotagmin, a putative calcium sensor for exocytosis, to a microdomain of calcium in£ux 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.