Biophysical Coupling Mechanisms in NMDA Receptor Gating

Abstract

NMDA receptors (NMDARs) are heterotetrameric glutamate-gated ion channels, typically composed of 2 GluN1 and 2 GluN2 subunits. The central function of NMDARs is gating - the process of converting agonist binding energy into pore opening by repositioning M3, the major pore-lining transmembrane helix. For glutamate receptors, it is largely assumed, though lacking direct evidence, that gating occurs through tension: ligand-binding domain (LBD) closure around an agonist generates tension in the linkers connecting the LBD to M3 (the M3-S2 linker), shifting M3 away from the central pore axis. To directly test this model, we altered the tension of the GluN1 and GluN2A M3-S2 linkers through residue insertions (to decrease tension) or deletions (to increase tension). Based on single-channel analyses, we find that these manipulations specifically affect pore opening (as opposed to ligand binding) and that they alter gating more dramatically in GluN2A than in GluN1. All-atom molecular dynamics simulations on a modeled GluN1/GluN2A receptor showed that this subunit-specific difference may arise, in part, from the disparate extensions and orientations of the GluN1 vs GluN2A M3-S2 linkers. Our functional data also suggests that the GluN1 M3-S2 linker gates primarily through tension. using rate equilibrium free energy relationship (REFER) analysis and length-tension analysis, we find that for the GluN1 M3-S2 linker, tension arises primarily during the C1-O1 transition (assuming a linear kinetic scheme) with a spring constant of ∼7.2 pN/nm, agreeing well with other biological springs. In contrast, our functional data suggests that mechanisms other than tension mainly mediate the role of the GluN2A M3-S2 linker in gating. Alternative mechanisms may include twisting and changes in electrostatic interactions.