Munc13-3 Is Required for the Developmental Localization of Ca²⁺ Channels to Active Zones and the Nanopositioning of Ca_v2.1 Near Release Sensors.

Spatial relationships between Ca_v 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_v2.1 and Ca_v2.2 channels, alters the localization of Ca_v2.1, and is required for the development of tight, nanodomain coupling at parallel-fiber AZs. We combined EGTA application and Ca²⁺-channel pharmacology in electrophysiological and two-photon Ca²⁺ imaging experiments with quantitative freeze-fracture immunoelectron microscopy and mathematical modeling. We found that a normally occurring developmental shift from release being dominated by Ca²⁺ influx through Ca_v2.1 and Ca_v2.2 channels with domain overlap and loose coupling (microdomains) to a nanodomain Ca_v2.1 to sensor coupling is impaired in Munc13-3-deficient synapses. Thus, at AZs lacking Munc13-3, release remained triggered by Ca_v2.1 and Ca_v2.2 microdomains, suggesting a critical role of Munc13-3 in the formation of release sites with calcium channel nanodomains.

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