Interaction of the synprint site of N-type Ca²⁺ channels with the C2B domain of synaptotagmin I

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 theirs 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.