Bassoon and the synaptic ribbon organize Ca²⁺ channels and vesicles to add release sites and promote refilling

At the presynaptic active zone, Ca²⁺ influx triggers fusion of synaptic vesicles. It is not well understood how Ca²⁺ channel clustering and synaptic vesicle docking are organized. Here, we studied structure and function of hair cell ribbon synapses following genetic disruption of the presynaptic scaffold protein Bassoon. Mutant synapses-mostly lacking the ribbon-showed a reduction in membrane-proximal vesicles, with ribbonless synapses affected more than ribbon-occupied synapses. Ca²⁺ channels were also fewer at mutant synapses and appeared in abnormally shaped clusters. Ribbon absence reduced Ca²⁺ channel numbers at mutant and wild-type synapses. Fast and sustained exocytosis was reduced, notwithstanding normal coupling of the remaining Ca²⁺ channels to exocytosis. In vitro recordings revealed a slight impairment of vesicle replenishment. Mechanistic modeling of the in vivo data independently supported morphological and functional in vitro findings. We conclude that Bassoon and the ribbon (1) create a large number of release sites by organizing Ca²⁺ channels and vesicles, and (2) promote vesicle replenishment.