DIRECT INTERACTION OF THE CALCIUM SENSOR PROTEIN SYNAPTOTAGMIN-I WITHA CYTOPLASMIC DOMAIN OF THE ALPHA(1)A SUBUNIT OF THE P Q-TYPE CALCIUM-CHANNEL/

Synaptotagmins are synaptic vesicle proteins containing two calcium-bi nding C2 domains which are involved in coupling calcium influx through voltage-gated channels to vesicle fusion and exocytosis of neurotrans mitters. The interaction of synaptotagmins with native P/Q-type calciu m channels was studied in solubilized synaptosomes from rat cerebellum , Antibodies against synaptotagmins I and II, but not TV co-immunoprec ipitated [I-125]omega-conotoxin MVIIC-labelled calcium channels, Direc t interactions were studied between in vitro-translated [S-35]synaptot agmin I and fusion proteins containing cytoplasmic loops of the alpha( 1)A subunit (BI isoform), Gel overlay revealed the association of syna ptotagmin I with a single region (residues 780-969) located in the int racellular loop connecting homologous domains II and III, Saturable ca lcium-independent binding occurred with equilibrium dissociation const ants of 70 nM and 340 nM at 4 degrees C and pH 7.4, and association wa s blocked by addition of excess recombinant synaptotagmin I, Direct sy naptotagmin binding to the pore-forming subunit of the P/Q-type channe l may optimally locate the calcium-binding sites that initiate exocyto sis within a zone of voltage-gated calcium entry.