A State-Specific Interaction Stabilizes NMDA Receptor Opening

Abstract

NMDA receptors mediate glutamate-gated excitatory currents that set the decay time of the excitatory post synaptic currents in the central nervous system. The current time course reflects a multi-step reaction mechanism whose structural underpinnings remain insufficiently understood. Generally, glutamate binding to extracellular domains (LBDs) initiates LBD constriction. The intramolecular movements culminate with the opening of the channel gate at the apex of M3 transmembrane helices. Several 3D arrangements for functional NMDA receptor constructs have been delineated in high resolution, however, their corresponding functional states and the order of their functional interconversion remain poorly delineated. We used all-atom targeted molecular dynamics simulations and previously reported NMDA receptor structures to identify pairs of residues whose state-dependent interactions appear to stabilize the channel gate in an open position. Here we present evidence for a functional interaction between one such pair, GluN1-I642 (on M3 helix) and GluN2A-L550 (on S1-M1 linker). We used single-molecule currents to delineate kinetic mechanisms for wild-type receptors and full length receptors with a series of single residue substitutions at these positions. Mutations at these sites significantly reduced channel open duration (wild-type 4.5 ± 0.7 ms; GluN1I642L 1.3 ± 0.3 ms; GluN2AL550I 1.9 ± 0.4 ms; GluN1I642L/GluN2AL550I 0.7 ± 0.1 ms) and burst open probability (wild-type 0.73 ± 0.05; GluN1I642L 0.08 ± 0.03; GluN2AL550I 0.08 ± 0.03; GluN1I642L/GluN2AL550I 0.003 ± 0.001). Results from double-mutant cycle analyses indicate strong coupling energy between the side chains of these proximally positioned residues and this interaction energy contributes ∼2.4 kJ to the stability of the open state. Overall, these data provide functional evidence for a new state-dependent interaction in NMDA receptors and suggest that absent this interaction, NMDA receptors generate currents with accelerated decay as observed for the human pathogenic variant GluN1-I642L.