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.
Mochida
S
S
Department of Physiology, Tokyo Medical College, Japan.
Fukuda
M
M
Niinobe
M
M
Kobayashi
H
H
Mikoshiba
K
K
D000109:Acetylcholine / Q000378:metabolism*; D000818:Animals; D000918:Antibody Specificity; D002135:Calcium-Binding Proteins; D002478:Cells, Cultured; D002799:Cholinergic Fibers / Q000737:chemistry / Q000378:metabolism; D004594:Electrophysiology; D015151:Immunoblotting; D007295:Inositol Phosphates / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D008564:Membrane Potentials / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D009474:Neurons / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D010833:Phytic Acid / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D013569:Synapses / Q000737:chemistry / Q000378:metabolism*; D050857:Synaptotagmins
D002135:Calcium-Binding Proteins; D007295:Inositol Phosphates; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; C081655:inositol-1,3,4,5-tetrakisphosphate; D050857:Synaptotagmins; C013571:inositol pentaphosphate; D010833:Phytic Acid; D000109:Acetylcholine