Munc13-3 Is Required for the Developmental Localization of Ca 2+ Channels to Active Zones and the Nanopositioning of Ca v 2.1 Near Release Sensors

Spatial relationships between Ca channels and release sensors at active zones (AZs) are a major determinant of synaptic fidelity. They are regulated developmentally, but the underlying molecular mechanisms are largely unclear. Here, we show that Munc13-3 regulates the density of Ca 2.1 and Ca 2.2 channels, alters the localization of Ca 2.1, and is required for the development of tight, nanodomain coupling at parallel-fiber AZs. We combined EGTA application and Ca -channel pharmacology in electrophysiological and two-photon Ca imaging experiments with quantitative freeze-fracture immunoelectron microscopy and mathematical modeling. We found that a normally occurring developmental shift from release being dominated by Ca influx through Ca 2.1 and Ca 2.2 channels with domain overlap and loose coupling (microdomains) to a nanodomain Ca 2.1 to sensor coupling is impaired in Munc13-3-deficient synapses. Thus, at AZs lacking Munc13-3, release remained triggered by Ca 2.1 and Ca 2.2 microdomains, suggesting a critical role of Munc13-3 in the formation of release sites with calcium channel nanodomains.