A Molecular Determinant of Subtype-Specific Desensitization in Ionotropic Glutamate Receptors

Abstract

AMPA and NMDA receptors are glutamate-gated ion channels that mediate fast excitatory synaptic transmission throughout the nervous system. In the continual presence of glutamate, AMPA and NMDA receptors containing the GluN2A or GluN2B subunit enter into a nonconducting, desensitized state that can impact synaptic responses and glutamate-mediated excitotoxicity. The process of desensitization is dramatically different between subtypes, but the basis for these differences is unknown. We generated an extensive sequence alignment of ionotropic glutamate receptors (iGluRs) from diverse animal phyla and identified a highly conserved motif, which we termed the "hydrophobic box," located at the extracellular interface of transmembrane helices. A single position in the hydrophobic box differed between mammalian AMPA and NMDA receptors. Surprisingly, we find that an NMDAR-to-AMPAR exchange mutation at this position in the rat GluN2A or GluN2B subunit had a dramatic and highly specific effect on NMDAR desensitization, making it AMPARlike. In contrast, a reverse exchange mutation in AMPARs had minimal effects on desensitization. These experiments highlight differences in desensitization between iGluR subtypes and the highly specific contribution of the GluN2 subunit to this process.

Molecular Mechanism of Disease-Associated Mutations in the Pre-M1 Helix of NMDA Receptors and Potential Rescue Pharmacology

“…Mutations of Pro to Ala, Gly, Ile, Leu as well as Gln increased the degree of desensitization relative to WT GluN2A, achieving steady-state levels that were only 20%, 29%, 29%, 1.5%, and 18% of the peak response, respectively, compared to 56% for WT GluN1/GluN2A (Fig 6D) (Table 7). This is consistent with a recent report showing strong effects on receptor desensitization by residues in the pre-M1 region [29]. NMDARs containing GluN2A-P552L showed no change in surface expression (S1 Fig), but underwent profound desensitization, showing greater than 98% reduction in current at steady-state (Fig 6E) (Table 7).…”

“…This region is conserved across all NMDAR GluN2 subunits [29] (Fig 2A) and is in van der Waals contact with the M3 helix (Fig 2B–2D). The proximity to the D2 domain that moves with agonist binding may allow the pre-M1 region to influence movement and position of the M3 helices that form the channel gate (Fig 2B–2D).…”

“…We further evaluated NMDAR functional changes caused by five GRIN missense mutations in the pre-M1 region of the receptor found in children with developmental delay, intellectual disability, and/or epilepsy (Table 2). Among these mutations, we focused on the mutant GluN2A-P552R, since this Pro residue is conserved across GluN subunits in multiple species (Fig 2; [29]). Our data show that GluN2A-P552R should be considered a gain-of-function variant because it shows increased glutamate / glycine potency and pronounced slowing of the glutamate current response time course, without concomitant changes in receptor surface expression or response amplitude.…”

“…Fourth, the pre-M1 helix and adjacent regions harbor structural determinants for subunit-selective positive allosteric modulators of the GluN2C- and GluN2D-containing NMDARs [56,57]. Fifth, mutations in and near pre-M1 can alter NMDAR function [14,29]. The position of the pre-M1 helix in the closed AMPAR and NMDAR structures [10,12,18] evokes the hypothesis that these helices, in van der Waals contact with the M3 gate, may serve to stabilize the gate in the closed conformation or otherwise impede M3 movement during channel opening.…”

Structural Bases of Desensitization in AMPA Receptor-Auxiliary Subunit Complexes

“…Typical 10–90% rise times were 200-300 μs, as measured from junction potentials at the open tip of the patch pipette after recordings. Since we recorded GluA2-mediated currents in whole-cell mode, the time constants of deactivation and desensitization were somewhat larger than the corresponding values measured using outside-out recordings (Alsaloum et al, 2016; Carbone and Plested, 2012; Robert et al, 2005; Weston et al, 2006; Yelshansky et al, 2004). Nevertheless, despite the apparent overestimation of the absolute values of these parameters, we believe that the differences in kinetics of the corresponding constructs are faithfully reflected in the data.…”