Distinct active zone protein machineries mediate Ca²⁺ channel clustering and vesicle priming at hippocampal synapses

Action potentials trigger neurotransmitter release at the presynaptic active zone with spatiotemporal precision. This is supported by protein machinery that mediates synaptic vesicle priming and clustering of Ca_V2 Ca²⁺ channels nearby. One model posits that scaffolding proteins directly tether vesicles to Ca_V2s; however, here we find that at mouse hippocampal synapses, Ca_V2 clustering and vesicle priming are executed by separate machineries. Ca_V2 nanoclusters are positioned at variable distances from those of the priming protein Munc13. The active zone organizer RIM anchors both proteins but distinct interaction motifs independently execute these functions. In transfected cells, Liprin-α and RIM form co-assemblies that are separate from Ca_V2-organizing complexes. At synapses, Liprin-α1-Liprin-α4 knockout impairs vesicle priming but not Ca_V2 clustering. The cell adhesion protein PTPσ recruits Liprin-α, RIM and Munc13 into priming complexes without co-clustering Ca_V2s. We conclude that active zones consist of distinct machineries to organize Ca_V2s and prime vesicles, and Liprin-α and PTPσ specifically support priming site assembly.