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.

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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).