Synaptic Ca2+ channels and neurexins are linked through direct and indirect binding complexes

Rapid release of neurotransmitters from presynaptic boutons is essential for brain function and is triggered by Ca²⁺ influx through voltage-gated calcium channels (VGCCs), which consist of an α1 pore-forming subunit, intracellular β subunits, and mostly extracellular α2δ auxiliary subunits. Neurexins (Nrxn) regulate neurotransmission and presynaptic Ca²⁺ influx, but the physical interactions with VGCC subunits remain unknown. Here, we examined these interactions in recombinant VGCC-Nrxn complexes using a nanobody-based co-precipitation system. We found that the α2δ-1 and α2δ-3 variants bind to the α1 pore-forming subunits of Ca_V2.1- and Ca_V2.2-type VGCCs with distinct preferences, whereas Nrxn1α and Nrxn1β do not directly interact with α1. Since Nrxn1α binds both α2δ variants but Nrxn1α/α2δ complexes do not include α1, mobile α2δ subunits may dynamically toggle between Nrxn1α and the Ca_V core. Additionally, Nrxn1α associates with α1 subunits independently of α2δ through the intracellular scaffold protein Mint2, which enhances Nrxn1α/α2δ complex formation by inhibiting full glycosylation of α2δ. Extracellularly shorter Nrxn1β cannot bind α2δ but can indirectly associate with Ca_V2 α1 pore-forming subunits via either Mint2 or CASK proteins. Therefore, our findings reveal distinct molecular complexes through which αNrxn and βNrxn variants interact with VGCC subunits to regulate presynaptic Ca²⁺ influx. (196 words).