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

g g-Conotoxin MVIIC binds to P/Q-type calcium channels with high affinity and N-type channels with low affinity. To reveal the residues essential for subtype selectivity, we synthesized Ala-scanning analogs of MVIIC. Binding assays using rat cerebellar P 2 membranes suggested that Thr 11 , Tyr 13 and Lys 2 are essential for binding to both N-and P/Q-type channels, whereas Lys 4 and Arg 22 are important for binding to P/Q-type channels. These results suggest that MVIIC interacts with P/Q-type channels via a large surface, in good agreement with previous observations using chimeric analogs.z 2000 Federation of European Biochemical Societies.

Recent studies suggested that autoantibodies that bind to voltage-dependent calcium channels and activate calcium entry may play a role in the progressive degeneration of motoneurons in sporadic amyotrophic lateral sclerosis. Immunoassays were performed to assess autoantibody titer in patients with amyotrophic lateral sclerosis or Lambert-Eaton myasthenic syndrome, a disease in which the presence of anti-calcium channel antibodies is well documented. Based on immunoprecipitation assays for antibodies against N-type calcium channels, only 8% (2/25) of amyotrophic lateral sclerosis patients had marginally positive titers, whereas 58% (18/31) of patients with Lambert-Eaton myasthenic syndrome had positive titers. Enzyme-linked immunosorbent assays with purified neuronal N-type calcium channels revealed immunoreactivity in 2 of 25 amyotrophic lateral sclerosis sera and 12 of 31 Lambert-Eaton myasthenic syndrome sera, which is not compatible with suggestions that enzyme-linked immunosorbent assay is a more sensitive technique for the detection of autoantibodies in amyotrophic lateral sclerosis. Furthermore, based on immunoprecipitation assays, amyotrophic lateral sclerosis sera were totally negative for antibodies against L-type calcium channels from skeletal muscle or brain. These data do not support the hypothesis that an autoimmune response against calcium channels plays a primary role in amyotrophic lateral sclerosis.