N-methyl-D-aspartate receptors are ionotropic glutamate receptors that are integral to synaptic transmission and plasticity. Variable GluN2 subunits in diheterotetrameric receptors with identical GluN1 subunits set very different functional properties, which support their individual physiological roles in the nervous system. To understand the conformational basis of this diversity, we assessed the conformation of the common GluN1 subunit in receptors with different GluN2 subunits using single-molecule fluorescence resonance energy transfer (smFRET). We established smFRET sensors in the ligand binding domain and modulatory amino-terminal domain to study an apo-like state and partially liganded activation intermediates, which have been elusive to structural analysis. Our results demonstrate a strong, subtype-specific influence of apo and glutamate-bound GluN2 subunits on GluN1 rearrangements, suggesting a conformational basis for the highly divergent levels of receptor activity, desensitization and agonist potency. Chimeric analysis reveals structural determinants that contribute to the subtype differences. Our study provides a framework for understanding GluN2-dependent functional properties and could open new avenues for subtype-specific modulation.

Current address Department of Molecular and Cellular Physiology Stanford University School of Medicine 279 Campus Drive Stanford CA 94305 USA

Current address DIANA Biotechnologies a s Průmyslová 596 252 50 Vestec Czech Republic

Department of Molecular and Cell Biology University of California Berkeley California 94720 USA

Helen Wills Neuroscience Institute University of California Berkeley California 94720 USA

Molecular Biology and Integrated Bioimaging Division Lawrence Berkeley National Laboratory Berkeley California 94720 USA

Weill Neurohub University of California Berkeley California 94720 USA

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