Subconductance states in NMDA receptor variants are Ca²⁺ impermeable and act at the M2 loop

A variety of de novo disease-associated variants are found in NMDA receptors (NMDARs), which carry out critical roles at synapses including mediating a Ca²⁺ influx. In the central permeation pathway, a vestibule is positioned between the external M3 gate and the M2 pore loop. A variant in the obligatory GluN1 subunit, Met641Ile, reduces Ca²⁺ permeability, but the mechanism of action is unknown. We find that other variants at GluN1-M641, including Leu, Val, and Thr, as well as variants at a homologous position in GluN2A (Val639Ile), do not alter Ca²⁺ permeability. Notably, and in contrast to the other variants, GluN1-Met641Ile has a prominent subconductance state. Molecular dynamics simulations show that the Ile641 side chain interacts more with the M2 loop, including the N-site in GluN1 and GluN2, than the wild-type Met641 side chain. N-site variants also induce subconductance states and reduce Ca²⁺ permeability. We conclude that subconductance states are largely Ca²⁺ impermeable and that the Met641Ile variant alters Ca²⁺ permeability by inducing a subconductance state via interacting with the M2 loop.