Changes of synaptotagmin interaction with t-SNARE proteins in vitro after calcium/calmodulin-dependent phosphorylation

The regulation of multiple phases of the life cycle of synaptic vesicles is carried out by a complex series of protein-protein interactions. According to the SNARE hypothesis the core of these interactions is a heterotrimeric complex formed by syntaxin, SNAP-25, and VAMP-synaptobrevin. Other protein s interacting with the core of the SNARE complex, such as voltage-activated calcium channels and synaptotagmin (a putative calcium sensor), are consid ered crucial for the calcium dependence of release and also molecular media tors of synaptic plasticity. Here the interaction of synaptotagmin with SNA RE proteins was studied in immunoprecipitated native complexes, and the eff ects of previous phosphorylation dephosphorylation on this interaction were analyzed. It is surprising that the interaction of synaptotagmin with synt axin and SNAP-25 in native complexes was not found to be calcium-dependent. However, previous incubation under dephosphorylating conditions decreased the synaptotagmin-syntaxin interaction. Stimulation of Ca2+/calmodulin-depe ndent protein kinase II, which endogenously phosphorylates synaptotagmin in synaptic vesicles, increased the interaction of syntaxin and SNAP-25 with synaptotagmin (particularly when measured in the presence of calcium), as w ell as increasing the binding of the kinase itself. These results suggest t hat calcium decreases synaptotagmin-t-SNARE interactions after dephosphoryl ation and increases them after phosphorylation. Overall, these results impl y a phosphorylation- dephosphorylation balance in regulation of the synapto tagmin-t-SNARE interaction and suggest a role for protein phosphorylation i n the modulation of calcium sensitivity in transmitter release.