Immunoglobulin G fractions from patients with Lambert-Eaton myasthenic syndrome (LEMS), an autoimmune disease of neuromuscular transmission, immunoprecipitate '2SI-labeled a-conotoxin GVIA-labeled calcium channels solubilized from rat brain. A 58-kDa antigen was detected by probing Western blots of partially purified calcium channels with LEMS plasma and IgG and was shown to be the relevant antigen in w-conotoxin receptor immunoprecipitation. Monoclonal antibody 1D12, produced by immunizing mice with synaptic membranes, has properties similar to these autoimmune IgGs in both immunoprecipitation and Western blotting assays. 1D12 antigen was purified by immunoaffinity chromatography and shown to bind LEMS IgG. The antigen was identified by screening a rat brain cDNA library with 1D12 and was found to have strong homology to the synaptic vesicle membrane protein synaptotagmin. Our results indicate therefore that these antibodies immunoprecipitate w-conotoxin receptors by binding to synaptotagmin that is associated with calcium channels. We suggest that the interaction between synaptotagmin and the voltage-gated calcium channel plays a role in docking synaptic vesicles at the plasma membrane prior to rapid neurotransmitter release and that autoantibody binding to a synaptotagmin-calcium-channel complex may be involved in the etiology of LEMS.

Synaptotagmin (p65) is an abundant synaptic vesicle protein of neurons and contains regions similar to the regulatory domain of protein kinase C. These domains are thought to be involved in calcium-dependent interaction with membrane phospholipids during exocytosis. To assess the functional role of synaptotagmin, synaptotagmin-deficient clonal variants of PC12 cells were isolated. All of the variant cells released catecholamine and adenosine triphosphate in response to elevated intracellular concentrations of calcium, which suggests that synaptotagmin is not essential for secretion of catecholamine and adenosine triphosphate from PC12 cells.

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

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.