Molecular definition of distinct active zone protein machineries for Ca²⁺ channel clustering and synaptic vesicle...

Action potentials trigger neurotransmitter release with minimal delay. Active zones mediate this temporal precision by co-organizing primed vesicles with Ca_V2 Ca²⁺ channels. The presumed model is that scaffolding proteins directly tether primed vesicles to Ca_V2s. We find that Ca_V2 clustering and vesicle priming are executed by separate machineries. At hippocampal synapses, 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 heterologous cells, Liprin-α and RIM from co-assemblies that are separate from Ca_V2-organizing complexes upon co-transfection. At synapses, Liprin-α1-4 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 of Ca_V2s. We conclude that active zones consist of distinct complexes to organize Ca_V2s and vesicle priming, and Liprin-α and PTPσ specifically support priming site assembly.