Rearrangements at the Heterodimer Interface are Integral to NMDA Receptor Activation

Abstract

Upon binding the neurotransmitter glutamate and the obligatory co-agonist glycine, NMDA receptors activate by opening a membrane permeable pore or desensitize by switching into a high-affinity non-conducting conformation. Both activation and desensitization require that the agonist-binding clamshell within each subunit closes to engulf the agonists. It has been hypothesized that this movement strains the contacts between agonist-binding domains of GluN1 and GluN2 and rupture of this interface causes receptor desensitization by disengaging agonist-binding from pore-opening. To investigate the role of inter-subunit contacts in NMDA receptor gating, we cross-linked the dimer interface by introducing cysteine residues at positions predicted to interact across subunits: N521 and L777 of GluN1 and E516 and L780 of GluN2A, respectively. Steady-state single-channel recordings indicated that cross-linked receptors had drastically reduced open probabilities (∼200-fold, Po = 0.0032) due to ∼5-fold shorter openings and ∼100-fold longer closures (means, SEM): MOT = 1.8 ± 0.2 ms, MCT = 792 ± 213 ms (n = 6; 80,028 events). However, the mean duration of closed intervals associated with desensitization remained unaltered (wt, tauD=2700 ms; mut, tauD=3150). Reduction of the disulfide bonds (10 mM DTT) significantly potentiated single channel currents (means, SEM: Po = 0.14 ± 0.02) by restoring the mean duration of openings (11.7 ± 1.6 ms) and significantly shortening mean closed durations (90 ± 19 ms), but had no discernible effects on microscopic desensitization (n = 7; 262,396 events). Based on these data, we propose that flexibility in the heterodimer interface at the level of agonist-binding domains represents an integral part of NMDA receptor activation.