The types of voltage-dependent calcium channels (VDCCs) present in the cholinergic terminals isolated from the electric organ of the ray, Narke japonica, were characterized on the basis of their pharmacological sensitivity to specific antagonists. Inhibition of these channel types by autoantibodies from patients with the Lambert-Eaton syndrome (LES) was then studied to determine the specificity of the pathogenic IgG. In normal untreated synaptosomal preparations, maximal doses of N- and P and/or Q-type Ca2+ channel antagonists, ω-conotoxin GVIA and ω-agatoxin IVA, inhibited depolarization-evoked ACh release by 47% and 43%, respectively. Calciseptine, an L-type VDCC antagonist, caused a 20% reduction in the release. This indicates that the exocytotic release process is predominantly mediated by N- and P/Q-type VDCCs. LES IgG or sera caused an inhibition of ACh release by 39-45% in comparison with the control antibody-treated preparations. The ionomycin-induced ACh release, however, was not altered by the antibodies. Additionally, the same LES antibodies inhibited whole-cell calcium currents (ICa) in bovine adrenal chromaffin cells. Thus, the pathogenic antibodies exert their action on VDCCs present in the synaptosomes. The efficacy of three Ca2+ channel antagonists in blocking ACh release was determined in preparations pretreated with LES IgG. ω-Agatoxin IVA produced only an additional 3-5% reduction in release beyond that obtained with LES antibodies. Despite the pretreatment with LES IgG, ω-conotoxin GVIA and calciseptine inhibited the release to nearly their control levels. These results indicate that LES antibodies mainly downregulate P/Q-type Ca2+ channels which contribute to presynaptic transmitter release from the cholinergic nerve terminals of electric organ. The present findings are consistent with the hypothesis that P/Q-type VDCCs at the neuromuscular junction are the target of LES antibodies and that their inhibition by the antibodies produces the characteristic neuromuscular defect in this disease. Lambert-Eaton syndrome (LES) is a presynaptic disorder characterized by impaired quantal release of acetylcholine (ACh) at the neuromuscular junction (Elmqvist & Lambert, 1968). LES is often associated with small-cell lung cancer (SCLC; O'Neill, Murray & Newsom-Davis, 1988), a neuroendocrine tumour that expresses neuronal voltage-dependent Ca2+ channels (VDCCs; Viglione, O'Shaughnessy & Kim, 1995). Current evidence supports the idea that the syndrome, which has an autoimmune origin, is a result of the inhibition of presynaptic VDCCs by the channel-specific autoantibodies (reviewed in Vincent, Lang & Newsom-Davis, 1989; Wray, 1990). Although the precise immunogen(s) has not yet been determined, an autoimmune response against tumour antigen(s) is thought to trigger the production of pathogenic autoantibodies (Roberts, Perera, Lang, Vincent & Newsom-Davis, 1985; Viglione et al. 1995). Morphological and electrophysiological studies collectively support the view that the LES antibodies downregulate the presynaptic Ca2+ channels mediating exocytotic release of ACh from the motor nerve terminal. In freeze-fracture electron micrographs of presynaptic membranes of the neuromuscular junctions of LES patients (Fukunaga, Engel, Osame & Lambert, 1982), active zone particles, hypothesized to be VDCCs, were spatially disorganized and fewer in number. Exposure of cultured neuronal and endocrine cells to IgG from LES patients causes a pronounced reduction in whole-cell currents carried by VDCCs (Kim & Neher, 1988; Peers, Lang, Newsom-Davis & Wray, 1990; Grassi, Magnelli, Carabelli, Sher & Carbone, 1994; Garcia & Beam, 1996; Meriney, Hulsizer, Lennon & Grinnell, 1996). Individual channel activation kinetics and conductance were unchanged, however, indicating that the pathogenic IgG acts to downregulate the number of functional channels (Kim & Neher, 1988; Grassi et al. 1994). LES antibodies also reduce depolarization-induced uptake of 45Ca2+ by SCLC cell lines (Roberts et al. 1985; De Aizpurua, Lambert, Griesman, Olivera & Lennon, 1988). As with antibodies against acetylcholine receptors (AChRs) in myasthenia gravis (MG), antigenic modulation and cross-linking of the channels are the events associated with pathogenic action of LES IgG (Peers, Johnston, Lang & Wray, 1993). Serological studies have also revealed the pathogenic interaction of LES autoantibodies with specific VDCCs. LES IgG can immunoprecipitate N-type VDCCs labelled with [125I]ω-conotoxin GVIA from chick and rat brain synaptosomes (Martin-Moutot, Lang, Newsom-Davis & Seager, 1995), human neuroblastoma cells (Sher et al. 1989) and SCLC cells (De Aizpurua et al. 1988). More recent work, however, indicates that 95% of LES patients possess antibodies which immunoprecipitate P and/or Q-type VDCCs labelled with [125I]ω-conotoxin MVIIC (Lennon et al. 1995). Despite overwhelming evidence for an autoimmune pathogenesis, no animal model of LES, such as that so well established for MG (Lindstrom, 1979), has been fully developed and characterized. In human myasthenia gravis, a postjunctional disorder of the skeletal neuromuscular junction, AChR proteins isolated from postsynaptic membranes of Torpedo electric organ have served as a highly valuable antigen in inducing the anti-AChR antibody production. In an attempt to produce a similar animal model by active immunization, we have recently immunized mice and rats with Narke japonica synaptosomes containing cholinergic presynaptic nerve terminals (Kim et al. 1998). Consistent with the presynaptic impairment characterizing LES, neuromuscular junctions in these immunized animals exhibit a reduction in the quantal content of nerve-evoked end-plate potentials (EPPs). In a previous study (Chapman, Rabinowitz, Korczyn & Michaelson, 1990), animals injected with similar cholinergic synaptosomes from Torpedo were also found to develop electromyographic signs of the syndrome. These studies, however, were not able to address which components of the synaptosome are involved in the autoimmune disease process. The absence of such information thus provides further impetus to identify and characterize the putative Ca2+ channel antigens contained in these electric ray synaptosomes. The aim of the present study is threefold. First is to characterize the types of Ca2+ channels present in the electric organ synaptosomes of the Japanese electric ray, Narke japonica. Second is to provide direct evidence that LES autoantibodies reduce the depolarization-induced ACh release from these synaptosomes. Third, we explored which types of Ca2+ channels are impaired by LES autoantibodies, thus suggesting the type(s) implicated in the disease process. Synaptosomes from electric organs used in the present work consist of pure cholinergic nerve terminals and are free from postsynaptic membranes. The ACh release characteristics in this preparation are similar to those at the neuromuscular junction (Dunant & Muller, 1986). Thus the results obtained using the synaptosomes are likely to be relevant to the neuromuscular junction. Furthermore, our ability to assess the reduction in ICa by LES antibodies in bovine adrenal chromaffin cells and to correlate this reduction with the inhibition of synaptosomal ACh release provides compelling evidence that LES antibodies interact with Ca2+ channels functionally coupled to the transmitter release. In addition to their value in the induction of experimental autoimmune Lambert-Eaton syndrome, cholinergic nerve terminals of electric organ appear to be an ideal system with which to assess directly the action of LES IgG on cholinergic transmitter release.