Synaptic targeting of N-type calcium channels in hippocampal neurons

N-type calcium (Ca²⁺) channels play a critical role in synaptic function, but the mechanisms responsible for their targeting in neurons are poorly understood. N-type channels are formed by an α_1B (Ca_v2.2) pore-forming subunit associated with β and α2δ auxiliary subunits. By expressing epitope-tagged recombinant α_1B subunits in rat hippocampal neuronal cultures, we demonstrate here that synaptic targeting of N-type channels depends on neuronal contacts and synapse formation. We also establish that the C-terminal 163 aa (2177-2339) of the α_1B-1 (Ca_v2.2a) splice variant contain sequences that are both necessary and sufficient for synaptic targeting. By site-directed mutagenesis, we demonstrate that postsynaptic density-95/discs large/zona occludens-1 and Src homology 3 domain-binding motifs located within this region of the α_1B subunit (Maximov et al., 1999) act as synergistic synaptic targeting signals. We also show that the recombinant modular adaptor proteins Mint1 and CASK colocalize with N-type channels in synapses. We found that the α_1B-2 (Ca_v2.2b) splice variant is restricted to soma and dendrites and postulated that somatodendritic and axonal/presynaptic isoforms of N-type channels are generated via alternative splicing of α_1B C termini. These data lead us to propose that during synaptogenesis, the α_1B-1 (Ca_v2.2a) splice variant of the N-type Ca²⁺ channel pore-forming subunit is recruited to presynaptic locations by means of interactions with modular adaptor proteins Mint1 and CASK. Our results provide a novel insight into the molecular mechanisms responsible for targeting of Ca²⁺ channels and other synaptic proteins in neurons.