Agonist binding to the NMDA receptor drives movement of its cytoplasmic domain without ion flow

Abstract

The NMDA receptor (R) plays important roles in brain physiology and pathology as an ion channel. Here we examine the ion flow-independent coupling of agonist to the NMDAR cytoplasmic domain (cd). We measure FRET between fluorescently tagged cytoplasmic domains of GluN1 subunits of NMDARs expressed in neurons. Different neuronal compartments display varying levels of FRET, consistent with different NMDARcd conformations. Agonist binding drives a rapid and transient ion flow-independent reduction in FRET between GluN1 subunits within individual NMDARs. Intracellular infusion of an antibody targeting the GluN1 cytoplasmic domain blocks agonist-driven FRET changes in the absence of ion flow, supporting agonist-driven movement of the NMDARcd. These studies indicate that extracellular ligand binding to the NMDAR can transmit conformational information into the cell in the absence of ion flow.

Keywords: FRET-FLIM; antibody infusion; conformational change; metabotropic; receptor cross-linking.

Fig. 1.

FRET between GluN1 subunits at individual NMDARs. (A) FLIM of dendrite and spines expressing indicated constructs (along with GluN2B in all figures). Pseudocolor (scale below) indicates GFP lifetime at each pixel. (Scale bar, 5 µm.) (B) Average GluN1-GFP lifetime for spines and corresponding dendritic segments (located under the spines) expressing indicated constructs; n > 20 neurons; > 400 spines (for each condition); ⁺⁺⁺P < 0.001 (Mann–Whitney). Error bars indicate SEM in all figures. (C) Model of intrareceptor FRET between GluN1-GFP and GluN1-mCherry in NMDAR; GluN2 cytoplasmic domain not shown for clarity (GluN2 N-terminal domain is dark blue). Two-photon excitation (brown), emission (green, red), and FRET (gray) energies indicated. (D) Plot of first vs. second mean lifetime measurement of GluN1-GFP/GluN1-mCherry/GluN2 expressing spines (gray circles) and dendrites (white circles with black outline). Black line is best fit minimizing both x and y distances (spines: full line; dendrites: dotted line); n = 309 spines, or dendritic segments, 17 neurons.

Fig. 2.

GluN1-GFP/GluN1-mCherry FRET occurs within individual NMDARs and is unaffected by induced receptor crosslinking. (A) Plot of lifetime vs. fluorescence intensity with best fit line (solid) and with interreceptor FRET expected line (dashed). Each data point is mean of all spines from one neuron expressing GluN1-GFP/GluN1-mCherry; n = 150 neurons (Materials and Methods). (B) Ligand-induced lifetime increase should be unaffected by extracellular cross-linking for intrareceptor FRET, but blocked for interreceptor FRET. (C) Representative fluorescence intensity images of neurons expressing GluN1-GFP/GluN1-mCherry in indicated conditions at indicated times before and after photobleaching spines encircled in red. (Scale bar, 5 µm.) (D) FRAP curves for indicated conditions; n >18 neurons; > 22 spines ⁺⁺⁺P < 0.001; ⁺⁺P < 0.01; ⁺P < 0.05; error bars, SEM. (E) Representative FLIM images of neurons in indicated conditions in 7CK. (Scale bar, 5 µm.) (F) Plot of FRET efficiency in spines with indicated treatments; n > 30 neurons; > 550 spines per condition.

Fig. 3.

Agonist binding induces conformational change in NMDAR cytoplasmic domain without ion flux. (A) Representative FLIM images of neurons expressing indicated constructs (with GluN2B in all figures) before and in 25 µM NMDA. (Scale bar, 5 µm.) Dendritic segments are masked for clarity (see SI Appendix, Fig. S3 for unmasked segments). (B) Average NMDA-induced spine GluN1-GFP lifetime change for indicated constructs and conditions, n > 20 neurons, > 495 spines for each condition; ⁺⁺⁺P < 0.001; error bars SEM; Mann–Whitney U test. (C) Model consistent with FRET changes in NMDAR.

Fig. 4.

NMDA-induced FRET changes are blocked by intracellular infusion of GluN1 C-ter antibody. (A) GluN1 C terminus (GluN1 Cter) antibody introduced in neurons expressing GluN1-GFP/GluN1-mCherry to restrain NMDARcd movement. (B) Representative images of a neuron infused with Alexa Fluor 488 conjugated GluN1 Cter Ab, along with red dye. [Scale bars, 10 μm (Left); 2 μm (Right).] (C) Representative FLIM images of neurons expressing GluN1-GFP/GluN1-mCherry in 7CK and indicated conditions, infused with indicated antibody 30–60 min before imaging. (Scale bar, 5 μm.) (D) Average spine GluN1-GFP lifetime change for conditions in C. N, CTRL Ab (16 neurons, 378 spines), GluN1 Cter Ab (21 neurons, 478 spines). ⁺⁺⁺P < 0.001; error bars SEM; unpaired t test.

Fig. 5.

Time course of NMDA-driven conformational changes of NMDAR cytoplasmic domain. (A) Representative FLIM images of neurons expressing indicated constructs before (Left) during and after (Right) NMDA application in 7CK (Materials and Methods). (Scale bar, 5 μm.) (B) Plot of GluN1-GFP spine lifetime change, relative to baseline, in 7CK (blue) or APV (black) before, during (orange bar) and after NMDA application; n > 300–600 spines, > 13 neurons per condition; ***P < 0.001 compared with baseline value (Wilcoxon); ⁺⁺⁺P < 0.001 compared with value in APV (Mann–Whitney). (C) Plot of change, relative to baseline, in GluN1-GFP lifetime induced by glutamate uncaging (orange bar). Blue, in 7CK; black, in APV; n > 235 spines, > 35 neurons per condition; ⁺P < 0.05, ⁺⁺P < 0.01, comparing values in 7CK and APV (Mann–Whitney); *P < 0.05, ***P < 0.001 compared with baseline value (Wilcoxon).