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. 2018 Nov 13;9(1):4769.
doi: 10.1038/s41467-018-07236-4.

Unmasking GluN1/GluN3A Excitatory Glycine NMDA Receptors

Affiliations
Free PMC article

Unmasking GluN1/GluN3A Excitatory Glycine NMDA Receptors

Teddy Grand et al. Nat Commun. .
Free PMC article

Abstract

GluN3A and GluN3B are glycine-binding subunits belonging to the NMDA receptor (NMDAR) family that can assemble with the GluN1 subunit to form unconventional receptors activated by glycine alone. Functional characterization of GluN1/GluN3 NMDARs has been difficult. Here, we uncover two modalities that have transformative properties on GluN1/GluN3A receptors. First, we identify a compound, CGP-78608, which greatly enhances GluN1/GluN3A responses, converting small and rapidly desensitizing currents into large and stable responses. Second, we show that an endogenous GluN3A disulfide bond endows GluN1/GluN3A receptors with distinct redox modulation, profoundly affecting agonist sensitivity and gating kinetics. Under reducing conditions, ambient glycine is sufficient to generate tonic receptor activation. Finally, using CGP-78608 on P8-P12 mouse hippocampal slices, we demonstrate that excitatory glycine GluN1/GluN3A NMDARs are functionally expressed in native neurons, at least in the juvenile brain. Our work opens new perspectives on the exploration of excitatory glycine receptors in brain function and development.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
The GluN1 antagonist CGP-78608 awakes GluN1/GluN3A receptors. a Pre-application of CGP-78608 massively potentiates excitatory glycine GluN1/GluN3A receptor responses. Bar graphs indicate effect on extent (Iss/Ipeak) and kinetics (τdes) of desensitization. ***P < 0.001, Student’s t-test, (n = 10); **P = 0.002, Mann–Whitney test (n = 10). b CGP-78608 potentiation dose-response curve obtained in presence of 100 µM glycine. Currents were measured at the peak. EC50 = 26.3 ± 5 nM, nH = 1.45 (n = 4–11). c Representative current traces showing the relative potentiation of various GluN1 antagonists compared to CGP-78608. Traces are normalized to the glycine-induced peak currents obtained prior to drug application. d Quantification of effects of GluN1 antagonists tested in c on peak and steady-state current levels (n = 4-6). N1-F484A refers to the GluN1-F484A mutant subunit. All recordings were performed in HEK293 cells. Data are mean ± SEM
Fig. 2
Fig. 2
Kinetics and activity-dependence of CGP-78608 potentiation at GluN1/GluN3A receptors. a CGP-78608 washout kinetics in the presence of continuous or pulses of glycine. CGP-78608 was applied at 500 nM. Bar graph: comparison of the CGP-78608 washout kinetics in the active (upper trace) or resting state (lower trace). n.s. P = 0.198, Student’s t-test; (n = 4-6). b CGP-78608 potentiation is state-dependent. Potentiation by CGP-78608 (500 nM) is greatly reduced when the compound is applied after, rather than before, application of glycine (pre-Gly vs post-Gly, respectively). Bar graph: potentiation measured at steady-state. **P = 0.008, Mann–Whitney test, (n = 5). Data are mean ± SEM
Fig. 3
Fig. 3
GluN1/GluN3A receptors display strong redox sensitivity. a Structure of the GluN1 and GluN3A ABDs (PDB 4KCC and 4KCD, respectively). Cysteines involved in endogenous redox-sensitive disulfide bridges are highlighted (sulfur atoms shown in yellow). The central cartoon illustrates domain organization in GluN1/GluN3A receptors; NTD, N-terminal domain; ABD, agonist-binding domain; TMD, transmembrane domain; CTD, C-terminal domain. b Effect of TCEP treatment (5 mM, 20 min) on wild-type and mutant GluN1/GluN3A receptors expressed in HEK 293 cells. Glycine was applied at 100 µM. Each trace comes from a separate cell. c Quantification of tail currents observed upon washout of glycine on wild-type and mutant GluN1/GluN3A receptors. Inset indicates current tags used for quantification. n.s. P = 0.125 and 0.337, Student’s t-test (n = 4–9). Note that non-treated receptors containing wild-type GluN3A subunits do not exhibit tail currents. Data are mean ± SEM. d Effect of reduced glutathione (GSH, 50 mM for 20 min) on wild-type GluN1/GluN3A receptors expressed in HEK293 cells. Glycine was applied at 100 µM. e Effect of TCEP treatment (5 mM, 20 min) on wild-type GluN1/GluN3A receptors expressed in Xenopus oocytes. The pair of current traces corresponds to responses before and after TCEP treatment on the same cell. Glycine was applied at 100 µM
Fig. 4
Fig. 4
High-glycine sensitivity and tonic activation of reduced GluN1/GluN3A receptors. a Glycine deactivation kinetics of wild-type and mutant receptors in presence of CGP-78608 (500 nM). Glycine was applied at 100 µM. b Quantification of glycine deactivation kinetics. *P = 0.027, Student’s t-test; ***P < 0.001, Student’s t-test (n = 4–7). Inset: representative current traces and overlaid fits following glycine washout. c Quantification of glycine-induced steady-state outward current shifts following TCEP treatment (see Text). Currents at steady-state (I1) are expressed as percentage of I0 current (see Fig. 2c). ***P < 0.001, Student’s t-test; n.s. P = 0.95 and 0.49, respectively, Student’s t-test; (n = 4–9). d Effect of CNQX (50 µM) on the holding current (current measured in the absence of glycine) for both wild-type and mutant GluN1-CS-CS/GluN3A-CS-CS receptors. e Effect of CNQX (50 µM) on basal and glycine-induced currents carried by GluN1-CS-CS/GluN3A-CS-CS mutant receptors. Note the strong inhibition by CNQX of the tail current (rebound current following glycine removal). Glycine was applied at 100 µM. All recordings were performed in HEK293 cells. Data are mean ± SEM
Fig. 5
Fig. 5
GluN1/GluN3A receptors are expressed and functional in juvenile hippocampal slices. a Schematic representation of the experimental protocol. Glycine (10 mM) is puffed onto voltage-clamped CA1 cells in acute hippocampal slices from young (P8-12) mice. b In control conditions, glycine puffs trigger very small inward currents in both wild-type (WT) and GluN3A-KO mice (upper black traces on both left and right). A typical response obtained in WT mice is shown at larger magnification and time scale in the top left inset. Bath application of CGP-78608 (CGP) leads to massive potentiation of glycine-elicited currents in WT mice, but has no effect in GluN3A-KO animals (bottom red traces). In WT mice, addition of DCKA (500 µM), a GluN1 and GluN3A glycine-binding site antagonist, eliminates currents obtained in the presence of CGP-78608 (bottom left green trace), thus further confirming that GluN1/GluN3A receptors mediate the responses to glycine. c Quantification of the experimental results obtained in panel b. WT mice, full bars; GluN3A-KO mice, empty bars. Data are illustrated as mean ± SEM
Fig. 6
Fig. 6
Schematic model of GluN1/GluN3A activation and modulation. For clarity, a single GluN1/GluN3A dimer is shown and the NTDs omitted. The upper scheme (a) shows receptor activation in control conditions. The GluN3A disulfide bridge conferring high redox sensitivity is highlighted in pink. The lower left scheme (b) illustrates activation sequence in presence of CGP-78608. Pre indicates application of CGP-78608 before glycine application while Post indicates the opposite. Pre-incubation with CGP-78608 prior to glycine application enhances receptor activity by preventing glycine binding to GluN1 and subsequent entry into desensitized states. The lower right scheme (c) illustrates activation sequence of reduced receptors (GluN3A ABD lower lobe disulfide bridge broken). The main modification is a large increase in the affinity of the GluN3A ABD for glycine resulting in tonic receptor activation by ambient glycine

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