biostudies-literatureTan CNIMHNICHD NIH HHSGiovanni Armenise-Harvard FoundationNIMH NIH HHSNINDS NIH HHSNational Institutes of HealthNINDSNational Science Foundation1498-1515.e8https://www.ebi.ac.uk/biostudies/studies/S-EPMC9081183biostudies-literatureUnknown110(9)Presynaptic active zones are molecular machines that control neurotransmitter secretion. They form sites for vesicle docking and priming and couple vesicles to Ca²⁺ entry for release triggering. The complexity of active zone machinery has made it challenging to determine its mechanisms in release. Simultaneous knockout of the active zone proteins RIM and ELKS disrupts active zone assembly, abolishes vesicle docking, and impairs release. We here rebuild docking, priming, and Ca²⁺ secretion coupling in these mutants without reinstating active zone networks. Re-expression of RIM zinc fingers recruited Munc13 to undocked vesicles and rendered the vesicles release competent. Action potential triggering of release was reconstituted by docking these primed vesicles to Ca<supNeuronRebuilding essential active zone functions within a synapse.PMC9081183R01MH113349R01NS083898P30NS072030P30 NS072030R01 MH113349R01 NS083898P50 HD105351DGE1144152Kaeser PSWang SSHTan Cde Nola GfalseRebuilding essential active zone functions within a synapse.Presynaptic active zones are molecular machines that control neurotransmitter secretion. They form sites for vesicle docking and priming and couple vesicles to Ca²⁺ entry for release triggering. The complexity of active zone machinery has made it challenging to determine its mechanisms in release. Simultaneous knockout of the active zone proteins RIM and ELKS disrupts active zone assembly, abolishes vesicle docking, and impairs release. We here rebuild docking, priming, and Ca²⁺ secretion coupling in these mutants without reinstating active zone networks. Re-expression of RIM zinc fingers recruited Munc13 to undocked vesicles and rendered the vesicles release competent. Action potential triggering of release was reconstituted by docking these primed vesicles to Ca<sup2022-01-01T00:00:00Z2022 May2026-05-09T19:46:02.672Z2025-04-04T03:41:51.585ZS-EPMC90811833517622110.1016/j.neuron.2022.01.026