Phosphorylation of the Synaptic Protein Interaction Site on N-type Calcium Channels Inhibits Interactions with SNARE Proteins

摘要: The synaptic protein interaction (synprint) site on the N-type calcium channel alpha1B subunit binds to soluble N-ethylmaleimide-sensitive attachment factor receptor (SNARE) proteins syntaxin and synaptosomal of 25 kDa (SNAP-25), this association may be required for efficient fast transmission. Protein kinase C (PKC) calmodulin-dependent type II (CaM KII) phosphorylated a recombinant his-tagged synprint polypeptide rapidly stoichiometry 3-4 mol phosphate/mol, whereas cAMP-dependent (PKA) cGMP-dependent (PKG) peptide more slowly <1 mol/mol. Two-dimensional phosphopeptide mapping revealed similar patterns phosphorylation polypeptides native rat brain subunits by PKC Cam KII. Phosphorylation with or CaM KII, but not PKA PKG, strongly inhibited binding SNAP-25, even at level free (15 microM) that stimulates maximal binding. In contrast, SNAP-25 KII did affect interactions site. Binding assays representing N- C-terminal halves indicate PKC- KII-mediated inhibition involves multiple, disperse sites. also its SNARE complexes containing SNAP-25. These results suggest serve as biochemical switch between channels complexes.