Role of the C2A domain of synaptotagmin in transmitter release as determined by specific antibody injection into the squid giant synapse preterminal.
Squid synaptotagmin (Syt) cDNA, including its open reading frame, was cloned and polyclonal antibodies were obtained in rabbits immunized with glutathione S-transferase (GST)-Syt-C2A. Binding assays indicated that the antibody, anti-Syt-C2A, recognized squid Syt and inhibited the Ca(2+)-dependent phospholipid binding to the C2A domain. This antibody, when injected into the preterminal at the squid giant synapse, blocked transmitter release in a manner similar to that previously reported for the presynaptic injection of members of the inositol high-polyphosphate series. The block was not accompanied by any change in the presynaptic action potential or the amplitude or voltage dependence of the presynaptic Ca2+ current. The postsynaptic potential was rather insensitive to repetitive presynaptic stimulation, indicating a direct effect of the antibody on the transmitter release system. Following block of transmitter release, confocal microscopical analysis of the preterminal junction injected with rhodamine-conjugated anti-Syt-C2A demonstrated fluorescent spots at the inner surface of the presynaptic plasmalemma next to the active zones. Structural analysis of the same preparations demonstrated an accumulation of synaptic vesicles corresponding in size and distribution to the fluorescent spots demonstrated confocally. Together with the finding that such antibody prevents Ca2+ binding to a specific receptor in the C2A domain, these results indicate that Ca2+ triggers transmitter release by activating the C2A domain of Syt. We conclude that the C2A domain is directly related to the fusion of synaptic vesicles that results in transmitter release.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins; D003001:Cloning, Molecular; D049832:Decapodiformes / Q000235:genetics; D004594:Electrophysiology; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D008954:Models, Biological; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D018377:Neurotransmitter Agents / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D017729:Presynaptic Terminals / Q000378:metabolism*; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D009435:Synaptic Transmission / Q000502:physiology*; D050857:Synaptotagmins
D002135:Calcium-Binding Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D018377:Neurotransmitter Agents; D010743:Phospholipids; D050857:Synaptotagmins; D002118:Calcium