Interaction of the Synprint Site of N-type Ca2+ Channels with the C2B Domain of Synaptotagmin I

• Published in: Proceedings of the National Academy of Sciences - PNAS 1997-05, Vol.94 (10), p.5405-5410
• Main Authors: Sheng, Zu-Hang, Yokoyama, Charles T., Catterall, William A.
• Format: Article
• Language: English
• Subjects: Antibodies; Binding Sites; Binding, Competitive; Biological Sciences; Calcium; Calcium - metabolism; Calcium Channels - chemistry; Calcium Channels - metabolism; Calcium Channels, L-Type; Calcium-Binding Proteins; Chromatography, Affinity; Drug interactions; Escherichia coli; Exocytosis; Glutathione Transferase; Immunoassay; Kinetics; Membrane Glycoproteins - chemistry; Membrane Glycoproteins - metabolism; Membrane Proteins - metabolism; Nerve Tissue Proteins - chemistry; Nerve Tissue Proteins - metabolism; Neurobiology; Neurology; Neurons; Peptide Fragments - metabolism; Peptides; Proteins; Qa-SNARE Proteins; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; Sequence Tagged Sites; Synaptic Transmission; Synaptosomal-Associated Protein 25; Synaptosomes; Synaptotagmin I; Synaptotagmins
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.94.10.5405

N-type Ca2+ channels mediate Ca2+ influx, which initiates fast exocytosis of neurotransmitters at synapses, and they interact directly with the SNARE proteins syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa) through a synaptic protein interaction (synprint) site in the intracellular loop connecting domains II and III of their α 1B subunits. Introduction of peptides containing the synprint site into presynaptic neurons reversibly inhibits synaptic transmission, confirming the importance of interactions with this site in synaptic transmission. Here we report a direct interaction of the synprint peptide from N-type Ca2+ channels with synaptotagmin I, an important Ca2+ sensor for exocytosis, as measured by an affinity-chromatography binding assay and a solid-phase immunoassay. This interaction is mediated by the second C2 domain (C2B) of synaptotagmin I, but is not regulated by Ca2+. Using both immobilized recombinant proteins and native presynaptic membrane proteins, we found that the synprint peptide and synaptotagmin competitively interact with syntaxin. This interaction is Ca2+-dependent because of the Ca2+ dependence of the interactions between syntaxin and these two proteins. These results provide a molecular basis for a physical link between Ca2+ channels and synaptotagmin, and suggest that N-type Ca2+ channels may undergo a complex series of Ca2+-dependent interactions with multiple presynaptic proteins during neurotransmission.