Synapse and Active Zone Assembly in the Absence of Presynaptic Ca²⁺ Channels and Ca²⁺ Entry

Presynaptic Ca_V2 channels are essential for Ca²⁺-triggered exocytosis. In addition, there are two competing models for their roles in synapse structure. First, Ca²⁺ channels or Ca²⁺ entry may control synapse assembly. Second, active zone proteins may scaffold Ca_V2s to presynaptic release sites, and synapse structure is Ca_V2 independent. Here, we ablated all three Ca_V2s using conditional knockout in cultured hippocampal neurons or at the calyx of Held, which abolished evoked exocytosis. Compellingly, synapse and active zone structure, vesicle docking, and transsynaptic nano-organization were unimpaired. Similarly, long-term blockade of action potentials and Ca²⁺ entry did not disrupt active zone assembly. Although Ca_V2 knockout impaired the localization of β subunits, α2δ-1 localized normally. Rescue with Ca_V2 restored exocytosis, and Ca_V2 active zone targeting depended on the intracellular C-terminus. We conclude that synapse assembly is independent of Ca_V2s or Ca²⁺ entry through them. Instead, active zone proteins recruit and anchor Ca_V2s via Ca_V2 C-termini.