N-type voltage-sensitive calcium channel interacts with syntaxin, synaptotagmin and SNAP-25 in a multiprotein complex

Expression of the N-type voltage sensitive calcium channel in Xenopus oocyt es along with syntaxin and p65 showed that the syntaxin-modified N-type cha nnel properties, were fully reversed by p65. The inward current was restore d to a significantly higher amplitude when all three proteins were present, suggesting that the channel interacts with syntaxin, p65 and SNAP-25 in a quaternary complex. Further support to a multicomplex formation between the channel and the synaptic proteins was drawn from the steady-state voltage inactivation profiles. A physical interaction of the N-type calcium channel with the vesicular protein synaptotagmin (p65) was demonstrated biochemica lly, using recombinant fusion proteins. The interaction is confined to a cy tosolic channel domain that separates segments II and III amino acids 710-1 090 of the N-type channel (N-LOOP710-1090) In vitro binding of recombinant N-LOOP710-1090 to p65 (amino acids 96-421) involves the two C2 domains of p 65, C2A domain [amino acids 96-265; p65(1-3)] and C2B domain [amino acids 2 45-421; p65(3-5)]. While the binding of C2A and C2B domains was calcium ind ependent, C2B domain binding to the N-Loop was inositol-hexaphosphate (IP6) -sensitive. The N-Loop(710-1090) binding to p65 was competed by syntaxin an d SNAP25, which are synaptic plasma membrane proteins. These combined funct ional and biochemical approaches provide evidence for a complex formation b etween the N-type channel and the exocytotic machinery which by generating fusion-competent vesicles map Function to regulate the process of synaptic secretion.