Role of the synprint site in presynaptic targeting of the calcium channel CaV2.2 in hippocampal neurons

Sequences in the cytoplasmic II–III loop of CaV2 voltage‐gated calcium channels, termed the synaptic protein interaction (synprint) site, are considered important for the functional incorporation of presynaptic calcium channels into the synaptic vesicle fusion apparatus. Two novel CaV2.2 splice variants lack large parts of the cytoplasmic II‐III loop (Δ1 R756‐L1139, Δ2 K737‐A1001) including the synprint protein–protein interaction domain. Here we expressed green fluorescent protein (GFP)‐α1B subunit fusion constructs of CaV2.2 splice variants in mouse hippocampal neurons to study their distribution in distinct neuronal compartments and to address the question of whether and how the synprint site functions in the presynaptic targeting of N‐type calcium channels. Similar to full‐length GFP‐α1B but divergent from the somatodendritic α1C‐HA (CaV1.2) channel type, the splice variants GFP‐α1B‐Δ1 and GFP‐α1B‐Δ2 were targeted into the axons. Nevertheless, their ability to form bona fide presynaptic clusters was almost abolished for GFP‐α1B‐Δ1 and significantly reduced for GFP‐α1B‐Δ2. Thus, the synprint site is important for normal synaptic targeting of CaV2.2 but not essential. Conversely, insertion of the synprint site into the II–III loop of α1C‐HA did not restore axonal targeting or synaptic clustering. Together these results indicate that protein–protein interactions with the synprint site must cooperate with other targeting mechanisms in the incorporation of CaV2.2 into presynaptic specializations of hippocampal neurons but are neither necessary nor sufficient for axonal targeting. The unique targeting properties of the splice variants lacking the synprint site are suggestive of specific functions of these calcium channels apart from activating fast synaptic transmission.