N-type Ca 2؉ channels mediate Ca 2؉ inf lux, 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 ␣ 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 Ca 2؉ channels with synaptotagmin I, an important Ca 2؉ 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 Ca 2؉ . 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 Ca 2؉ -dependent because of the Ca 2؉ dependence of the interactions between syntaxin and these two proteins. These results provide a molecular basis for a physical link between Ca 2؉ channels and synaptotagmin, and suggest that N-type Ca 2؉ channels may undergo a complex series of Ca 2؉ -dependent interactions with multiple presynaptic proteins during neurotransmission.During synaptic transmission, exocytosis of synaptic vesicles in neurons is initiated by the rapid influx of Ca 2ϩ through voltage-gated Ca 2ϩ channels within 200 sec. Major progress has been made toward the understanding of the molecular mechanisms that underlie Ca 2ϩ -dependent exocytosis by identifying proteins that are involved in this process and analyzing their interactions (1, 2). Vesicle docking and fusion are mediated by a core complex of proteins including the synaptic vesicle protein VAMP͞synaptobrevin (3) and the plasmalemmal proteins syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) (4-11). The synaptic vesicle protein p65 or synaptotagmin (12) binds Ca 2ϩ (13,14) and interacts with syntaxin in a Ca 2ϩ -dependent manner (15,16). It is thought to serve as a Ca 2ϩ sensor for fast, Ca 2ϩ -dependent neurotransmitter release (17)(18)(19)(20)(21)(22).N-type Ca 2ϩ channels are localized in nerve terminals (23-25) and participate in neurotransmitter release in central and peripheral synapses (26-28). They are coimmunoprecipitated and copurified with proteins of the synaptic core complex in immunochemical studies (4,5,29), and they are closely associated with sites of transmitter release in physiological experiments (30). The ␣ 1B subunits of N-type Ca 2ϩ channels (31, 32) interact with the synaptic core complex through a synaptic protein interaction (synprint) site in the intracellular loop connecting domains II and III (33, 34) in a Ca 2ϩ -dependent manner with maximum binding in the range of 10-30 M (35). Peptides containing th...