Optical profiling of NMDA receptor molecular diversity at synaptic and extrasynaptic sites

NMDA receptors (NMDARs) are glutamate-gated ion channels that play essential roles in brain development and function. NMDARs exist as multiple subtypes that differ in their subunit composition, distribution and signaling properties, with GluN1/GluN2A (GluN2A diheteromers), GluN1/GluN2B (GluN2B diheteromers) and GluN1/2A/2B (GluN2A/2B triheteromers) receptors prevailing. Studying these subtypes separately has proved difficult due to the limited specificity of available pharmacological and genetic approaches. Here, we designed a photoswitchable tool (Opto2B) enabling specific and reversible modulation of GluN2B diheteromers (while other receptor subtypes remain unaffected). Using Opto2B, we were able to establish the differential contribution of GluN2B diheteromers relative to GluN2A-receptors (GluN2A diheteromers and GluN2A/2B triheteromers) to synaptic and extrasynaptic NMDAR pools. In young postnatal CA1 hippocampal pyramidal cells, extrasynaptic NMDARs are exclusively composed of GluN2B diheteromers, whereas GluN2A subunits already populate synaptic sites. In adult CA1 cells, GluN2A-receptors predominate at both sites, with no preferential contribution of GluN2B diheteromers to extrasynaptic currents. Our study clarifies decades of controversial research and paves the way for interrogating NMDAR signaling diversity with unprecedented molecular and spatio-temporal resolution.