Emperador-Melero, J.; Andersen, J. W.; Metzbower, S. R.; Levy, A. D.; Dharmasri, P. A.; de Nola, G.; Blanpied, T. A.; Kaeser, P. S.

Action potentials trigger neurotransmitter release with minimal delay. Active zones mediate this temporal precision by co-organizing primed vesicles with CaV2 Ca2+ channels. The presumed model is that scaffolding proteins directly tether primed vesicles to CaV2s. We find that CaV2 clustering and vesicle priming are executed by separate machineries. At hippocampal synapses, CaV2 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-alpha and RIM from co-assemblies that are separate from CaV2-organizing complexes upon co-transfection. At synapses, Liprin-alpha1-4 knockout impairs vesicle priming, but not CaV2 clustering. The cell adhesion protein PTPsigma recruits Liprin-alpha, RIM and Munc13 into priming complexes without co-clustering of CaV2s. We conclude that active zones consist of distinct complexes to organize CaV2s and vesicle priming, and Liprin-alpha and PTPsigma specifically support priming site assembly.