Roles of synaptotagmin C2 domains in neurotransmitter secretion and inositol high-polyphosphate binding at mammalian cholinergic synapses.

To determine the functional role of synaptotagmin (Syt) regulatory domains, affinity-purified antibodies specific for C2A or C2B domains were injected into presynaptic neurons of cholinergic synapses formed between rat sympathetic neurons in culture. Following injection of anti-C2A antibody, postsynaptic responses evoked by presynaptic action potentials at a frequency of 0.05 Hz decreased rapidly, while anti-C2B antibody slowly decreased synaptic transmitter release. The inhibitory effect of anti-C2B antibody depended on the amount of synaptic activity. Asynchronous release induced by hypertonic solution was also affected by the antibodies. Anti-C2A antibody showed a dual action on miniature excitatory postsynaptic potentials, a decrease and following increase in the frequency, while synapses loaded with anti-C2B antibody showed a decrease in the frequency after long repetitive stimulation (0.05 Hz for more than 60 min). Anti-C2B antibody prevented the inhibition of acetylcholine release induced by injection of inositol 1,3,4,5-tetrakisphosphate (IP4), indicating that C2B domain may down-regulate transmitter release by IP4 binding. These results confirm similar experiments in the glutamatergic squid giant synapses and suggest a model in which Syt C2A and C2B domains differentially control synaptic vesicle trafficking in mammalian cholinergic terminals; C2A domain may act on the fusion step as a calcium sensor in synaptic vesicle exocytosis evoked by action potentials in addition to controlling spontaneous transmitter release, while C2B domain is involved in exo- and endocytosis.