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Changes of Synaptotagmin Interaction with t‐SNARE Proteins In Vitro After Calcium/Calmodulin‐Dependent Phosphorylation
Author(s) -
Verona Marina,
Zanotti Simona,
Schäfer Theo,
Racagni Giorgio,
Popoli Maurizio
Publication year - 2000
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.2000.0740209.x
Subject(s) - synaptotagmin 1 , syntaxin , microbiology and biotechnology , stx1a , phosphorylation , chemistry , synaptotagmin i , biology , snare complex , synaptic vesicle , biophysics , biochemistry , membrane protein , vesicle , membrane
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 proteins interacting with the core of the SNARE complex, such as voltage‐activated calcium channels and synaptotagmin (a putative calcium sensor), are considered crucial for the calcium dependence of release and also molecular mediators of synaptic plasticity. Here the interaction of synaptotagmin with SNARE proteins was studied in immunoprecipitated native complexes, and the effects of previous phosphorylation‐dephosphorylation on this interaction were analyzed. It is surprising that the interaction of synaptotagmin with syntaxin 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 Ca 2+ /calmodulin‐dependent 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 well as increasing the binding of the kinase itself. These results suggest that calcium decreases synaptotagmin‐t‐SNARE interactions after dephosphorylation and increases them after phosphorylation. Overall, these results imply a phosphorylation‐dephosphorylation balance in regulation of the synaptotagmin‐t‐SNARE interaction and suggest a role for protein phosphorylation in the modulation of calcium sensitivity in transmitter release.