Active zone scaffolds differentially accumulate Unc13 isoforms to tune Ca2+ channel–vesicle coupling

Coupling distances between synaptic vesicles and Ca 2+ channels determine the efficacy of neurotransmission. Böhme et al . find that presynaptic scaffold complexes spatiotemporally control Unc13 isoforms to establish two independent release pathways at subsynaptic active zones: Unc13B defines nascent, loosely coupled synapses whereas Unc13A facilitates release at mature synapses by tight coupling between Ca 2+ channels and synaptic vesicles. Brain function relies on fast and precisely timed synaptic vesicle (SV) release at active zones (AZs). Efficacy of SV release depends on distance from SV to Ca 2+ channel, but molecular mechanisms controlling this are unknown. Here we found that distances can be defined by targeting two unc-13 (Unc13) isoforms to presynaptic AZ subdomains. Super-resolution and intravital imaging of developing Drosophila melanogaster glutamatergic synapses revealed that the Unc13B isoform was recruited to nascent AZs by the scaffolding proteins Syd-1 and Liprin-α, and Unc13A was positioned by Bruchpilot and Rim-binding protein complexes at maturing AZs. Unc13B localized 120 nm away from Ca 2+ channels, whereas Unc13A localized only 70 nm away and was responsible for docking SVs at this distance. Unc13A null mutants suffered from inefficient, delayed and EGTA-supersensitive release. Mathematical modeling suggested that synapses normally operate via two independent release pathways differentially positioned by either isoform. We identified isoform-specific Unc13-AZ scaffold interactions regulating SV-Ca 2+ -channel topology whose developmental tightening optimizes synaptic transmission.