Molecular definition of distinct active zone protein machineries for Ca 2+ channel clustering and synaptic vesicle priming.

Action potentials trigger neurotransmitter release with minimal delay. Active zones mediate this temporal precision by co-organizing primed vesicles with Ca V 2 Ca 2+ channels. The presumed model is that scaffolding proteins directly tether primed vesicles to Ca V 2s. We find that Ca V 2 clustering and vesicle priming are executed by separate machineries. At hippocampal synapses, Ca V 2 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 V 2-organizing complexes upon co-transfection. At synapses, Liprin-α1-4 knockout impairs vesicle priming, but not Ca V 2 clustering. The cell adhesion protein PTPσ recruits Liprin-α, RIM and Munc13 into priming complexes without co- clustering of Ca V 2s. We conclude that active zones consist of distinct complexes to organize Ca V 2s and vesicle priming, and Liprin-α and PTPσ specifically support priming site assembly.