Trans-synaptic molecular context of NMDA receptor nanodomains

Tight coordination of the spatial relationships between protein complexes is required for cellular function. In neuronal synapses, many proteins responsible for neurotransmission organize into subsynaptic nanoclusters whose trans-cellular alignment modulates synaptic signal propagation. However, the spatial relationships between these proteins and NMDA receptors (NMDARs), which are required for learning and memory, remain undefined. Here, we mapped the relationship of key NMDAR subunits to reference proteins in the active zone and postsynaptic density using multiplexed super-resolution DNA-PAINT microscopy. GluN2A and GluN2B subunits formed nanoclusters with diverse configurations that, surprisingly, were not localized near presynaptic vesicle release sites marked by Munc13-1. Despite this, we found a subset of release sites was enriched with NMDARs, and modeling of glutamate release and receptor activation in measured synapses indicated this nanotopography promotes NMDAR activation. This subset of release sites was internally denser with Munc13-1, aligned with abundant PSD-95, and associated closely with specific NMDAR nanodomains. Further, NMDAR activation drove rapid reorganization of this release site/receptor relationship, suggesting a structural mechanism for tuning NMDAR-mediated synaptic transmission. This work reveals a new principle regulating NMDAR signaling and suggests that synaptic functional architecture depends on the assembly of and trans-cellular spatial relationships between multiprotein nanodomains.