Mechanism of SNARE protein binding and regulation of Cav2 channels by phosphorylation of the synaptic protein interaction site

Cav2.1 and Cav2.2 channels conduct P/Q-type and N-type Ca2+ currents that initiate neurotransmission and bind SNARE proteins through a synaptic protein interaction (synprint) site. PKC and CaMKII phosphorylate the synprint site and inhibit SNARE protein binding in vitro. Here we identify two separate microdomains that each bind syntaxin 1A and SNAP-25 in vitro and are regulated by PKC phosphorylation at serines 774 and 898 and CaMKII phosphorylation at serines 784 and 896. Activation of PKC resulted in its recruitment to and phosphorylation of CaV2.2 channels, but PKC phosphorylation did not dissociate CaV2.2 channel/syntaxin 1A complexes. Chimeric CaV2.1a channels containing the synprint site of Cav2.2 gain modulation by syntaxin 1A, which is blocked by PKC phosphorylation at the sites identified above. Our results support a bipartite model for the synprint site in which each SNARE-binding microdomain is controlled by a separate PKC and CaMKII phosphorylation site that regulates channel modulation by SNARE proteins.