Cryo-EM snapshots of NMDA receptor activation illuminate sequential rearrangements

Canonical W-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 (Po) of NMDARs composed of GluN1 and GluN2A subunits, together with judiciously chosen mutants and ligands, to achieve conditions in which receptors had a Po near unity. Using single-particle cryo-electron microscopy (cryo-EM), we captured three activated receptor states, each with distinct conformations of the gateforming 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 poreforming 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.