Heteromerization of ligand binding domains of N-methyl-D-aspartate receptor requires both coagonists, L-glutamate and glycine

Abstract

NMDA receptors (NMDAR) are voltage- and glutamate-gated heteromeric ion channels found at excitatory neuronal synapses, the functions of which are to mediate the mechanisms of brain plasticity and, thereby, its higher order functions. In addition to Glu, the activation of these heteromeric receptors requires Gly or d-Ser as a coagonist. However, it is not fully known as to why coagonism is required for the opening of NMDAR ion channels. We show herein that the ligand binding domains (LBD) of the GluN1 and GluN2A subunits of the NMDAR heterodimerize only when both coagonists, Glu and Gly/d-Ser, bind to their respective sites on GluN2 and GluN1. In the agonist-free state, these domains form homomeric interactions, which are disrupted by binding of their respective agonists. Also, in a heteromer formed by the LBDs, GluN2A is more sensitized to bind Glu, while the affinity of Gly for GluN1 remains unchanged. We thus provide direct evidence to show that coagonism is necessary for heteromeric pairing of LBDs, which is an essential step in forming functional ion channels in NMDARs.

Figure 1

Self-association of LBDs is disrupted by agonist binding. (A) c(M) distribution overlay after sedimentation velocity analysis of a primarily dimeric sample of GluN2A to highlight the increase in monomer population in the presence of l-Glu. Inset, SDS-PAGE showing purified protein preparations of GluN1-LBD and GluN2A-LBD respectively. (B) Sedimentation equilibrium analysis of GluN1 analyzed using SEDPHAT and fitted with the Species Analysis model for a combination of corresponding GluN1 monomeric and dimeric molecular weights. The bottom panel shows the residuals of the fitted data. (C) Sedimentation equilibrium data of GluN1 + 400 μM Gly were analyzed with SEDPHAT and fitted for GluN1 monomer size using the Species Analysis model. (D) Sedimentation velocity analysis of the GluN1-LBD analyzed using SEDFIT and showing c(M) distribution of molecular masses corresponding to monomeric and dimeric species in the presence or absence of Gly.

Figure 2

Heteromerization of GluN1 and GluN2A LBDs requires both coagonists. (A) Sedimentation velocity data analyzed using SEDFIT and showing the c(M) distribution of molecular masses corresponding to monomeric and dimeric species for the combination of GluN1 + GluN2A in the presence or absence of coagonists. (B) Bar diagrams showing the ratio of percent fraction of dimer/monomer species as in (A), quantified by the peak integration method in SEDFIT. The binding of coagonists leads to a higher ratio than that in the absence of ligands, l-Glu alone or Gly alone. * denotes p = 0.02, 0.03, and 0.02 respectively obtained from unpaired t-test analyses from two independent experimental sets. (C) c(M) distribution overlay to highlight the increase in dimer population in the presence of l-Glu/Gly or l-Glu/d-Ser combinations, but not in the presence of d-Ser alone. (D) Bar diagrams showing the ratio percent of dimer/monomer species as in (C), quantified by the peak integration method in SEDFIT. ** denotes p = 0.003 obtained from unpaired t-test analyses.

Figure 3

Heterodimerization of GluN1 and GluN2 requires both coagonists. (A, B) SEDPHAT analysis of GluN1/GluN2A in the presence of both l-Glu and Gly (A) or l-Glu and d-Ser (B) fitted to monomer and dimer molar masses using a combined average molecular weight of GluN1 and GluN2A of 36 000. (C) c(M) distribution overlay showing attenuation of dimerization in the presence of DCKA compared to that in the presence of l-Glu/Gly.

Figure 4

Binding of coagonists to NMDAR subunits. (A) ³H-l-Glu binding profile of GluN2A-LBD measured by equilibrium dialysis. (B) ³H-Gly binding profile of the GluN1-LBD. (C) Combination assay using both GluN1-LBD and GluN2A-LBD for ³H-l-Glu binding in comparison with GluN2A alone. (D) Combination assay using both GluN1-LBD and GluN2A-LBD for [³H]-Gly binding in comparison with GluN1 alone.

Figure 5

Binding of l-Glu to GluN2A is enhanced by Gly binding to GluN1. (A) [³H]-l-Glu equilibrium binding assay using equilibrium dialysis performed for the combination of GluN2A and GluN1 in the presence or absence of Gly. (B) Averaged B_max values from (A) are represented as bar graphs. (C) Combination assays using both GluN1 and GluN2A for [³H]-Gly binding in the presence or absence of l-Glu. (D) Averaged B_max values from (C) represented as bar graphs. No significant differences were observed for [³H]-Gly binding in the presence or absence of l-Glu. The K_D and B_max values obtained from the assays are summarized in Table 1. *** denotes p = 0.0002 obtained from unpaired t test analysis, n = 3.

Figure 6

Enhanced NMDAR channel conductance follows coagonist preincubation. (A) Whole cell recordings of l-Glu/Gly induced currents before and after preincubation with l-Glu. (B) Traces for l-Glu/Gly currents before and after Gly preincubation. (C) Traces for l-Glu/d-Ser currents before and after d-Ser preincubation. Each trace from (A–C) represents at least three recordings. Each inset shows overlay of traces with (red) or without (black) preincubation.

Figure 7

Comparison of NMDA-induced current peak amplitudes, as well as the area under the current peak between the control and coagonist preincubated samples. (A, B) Gly preincubation, (C, D) d-Ser preincubation, (E, F) l-Glu preincubation. p values obtained from paired t-tests in each case are shown.

Figure 8

Proposed mechanism of coagonist regulation in NMDAR activation. A model showing the possible mechanism of partial agonism for NMDAR activation based on homomeric and heteromeric interactions in the LBDs. In the absence of agonists the diagonally placed LBDs will tend to form homomeric interactions resulting in the closing of the ion channel pore. Binding of one of the agonists can disrupt the homomeric interaction of that subunit. Binding of the second coagonist will initiate heteromeric interactions. The agonist induced heteromeric interactions between LBDs causes realignment that could transduce down leading to channel opening. Inability of a single coagonist to induce domain realignments maintains the channel in the closed state. The red arrow in bold indicates that binding of l-Glu is favored by a Gly bound GluN1 as per our data. The red dotted arrow indicates that the dissociation rate of l-Glu is reduced when Gly remains bound to GluN1.