Cryo-EM snapshots of NMDA receptor activation illuminate sequential rearrangements

Canonical <i>N</i>-methyl-d-aspartate receptors (NMDARs) are glutamate-gated ion channels with critical roles in the development and function of the nervous system. The excitatory currents they produce reflect stochastic transitions between multiple agonist-bound closed- and open-pore states. We leveraged the intrinsically high open probability (<i>P</i>_o) of NMDARs composed of GluN1 and GluN2A subunits, together with judiciously chosen mutants and ligands, to achieve conditions in which receptors had a <i>P</i>_o near unity. Using single-particle cryo-electron microscopy (cryo-EM), we captured three activated receptor states, each with distinct conformations of the gate-forming M3 helices. Separately, we carried out single-channel electrophysiology, together with statistical modeling, to relate the cryo-EM structures to the gating reaction. NMDAR channel opening involves bending of the pore-forming M3 helices to produce a transient open-channel conformation, subsequently stabilized by new interactions between the D2-M3 linkers with the pre-M1 helices and the pre-M4 loops, to yield the stable open channel.