Direct interaction of the calcium sensor protein synaptotagmin I with a cytoplasmic domain of the α1A subunit of the P/Q‐type calcium channel

Synaptotagmins are synaptic vesicle proteins containing two calcium‐binding C2 domains which are involved in coupling calcium influx through voltage‐gated channels to vesicle fusion and exocytosis of neurotransmitters. The interaction of synaptotagmins with native P/Q‐type calcium channels was studied in solubilized synaptosomes from rat cerebellum. Antibodies against synaptotagmins I and II, but not IV co‐immunoprecipitated [125I]ω‐conotoxin MVIIC‐labelled calcium channels. Direct interactions were studied between in vitro‐translated [35S]synaptotagmin I and fusion proteins containing cytoplasmic loops of the α1A subunit (BI isoform). Gel overlay revealed the association of synaptotagmin I with a single region (residues 780–969) located in the intracellular loop connecting homologous domains II and III. Saturable calcium‐independent binding occurred with equilibrium dissociation constants of 70 nM and 340 nM at 4°C and pH 7.4, and association was blocked by addition of excess recombinant synaptotagmin I. Direct synaptotagmin binding to the pore‐forming subunit of the P/Q‐type channel may optimally locate the calcium‐binding sites that initiate exocytosis within a zone of voltage‐gated calcium entry.