Subunit arrangement in N-methyl-D-aspartate (NMDA) receptors

Abstract

N-Methyl-d-aspartate (NMDA) receptors, the main mediators of excitatory synaptic transmission, are heterotetrameric receptors. Typically, glycine binding NR1 subunits co-assemble with glutamate binding NR2 subunits to form a functional receptor. Here we have used luminescence resonance energy transfer (LRET) investigations to establish the specific configuration in which these subunits assemble to form the functional tetramer and show that the dimer of dimers structure is formed by the NR1 subunits assembling diagonally to each other. The distances measured by LRET are consistent with the NMDA structure predicted based on cross-linking investigations and on the structure of the full-length alpha-amino-5-methyl-3-hydroxy-4-isoxazole propionic acid (AMPA) receptor structure (1). Additionally, the LRET distances between the NR1 and NR2A subunits within a dimer measured in the desensitized state of the receptor are longer than the distances in the previously published crystal structure of the isolated ligand binding domain of NR1-NR2A. Because the dimer interface in the isolated ligand binding domain crystallizes in the open channel structure, the longer LRET distances would be consistent with the decoupling of the dimer interface in the desensitized state. This is similar to what has been previously observed for the AMPA subtype of the ionotropic glutamate receptors, suggesting a similar mechanism for desensitization in the two subtypes of the glutamate receptor.

FIGURE 1.

Possible arrangements of NR1-NR2A receptor agonist binding domains 1 and 2 based on full-length AMPA receptor crystal structure. The position of the NR1 subunit on the AMPA receptor structure is colored in red for domain 1 and in pink for domain 2. The position of NR2A subunit on the AMPA receptor structure is colored in green for domain 1 and in pale green for domain 2. Homologous sites tagged in the modified ΔNR1* and ΔNR2A* subunit for LRET measurements are highlighted.

FIGURE 2.

A, LRET lifetimes for ΔNR1*Thr-396Histag/ΔNR1*T396C-Th:ΔNR2A* labeled with terbium chelate:(Ni-NTA)₂Cy3 as measured by the sensitized emission of acceptor at 575 nm in the presence of saturating concentrations of agonists. No LRET signal was observed. B, LRET lifetimes for ΔNR1*:ΔNR2A*Asn-404Histag/ΔNR2A*N404C-Th labeled with terbium chelate:(Ni-NTA)₂Cy3 as measured by the sensitized emission of acceptor at 575 nm with saturating concentrations of agonists. C, LRET lifetimes measured at 575 nm for ΔNR1*Thr-396Histag:ΔNR2A* N404C-Th labeled with terbium chelate:(Ni-NTA)₂Cy3 under saturating concentrations of agonists. D, LRET lifetimes measured at 515 nm for ΔNR1*Th-525C-Th:ΔNR2A* labeled with terbium chelate:fluorescein at saturating concentrations of agonists. For all mutants, the sensitized lifetime shown is the difference between the lifetimes obtained before and after thrombin digestion. The residuals for the lifetime fits are shown below for each measurement (the y axis is in linear scale).

FIGURE 3.

Sites that were tagged in the modified ΔNR1* and ΔNR2A* subunit for LRET measurements within the dimer are highlighted in the crystal structure of NR1-NR2A dimer.

FIGURE 4.

A, LRET lifetimes for ΔNR1*775tetrahistag:ΔNR2A*S519C labeled with terbium chelate before (Donor only) and after the addition of Ni²⁺. B, LRET lifetimes for ΔNR1*525tetrahistag:ΔNR2A*L777C labeled with terbium chelate before (Donor only) and after the addition of Ni²⁺. The residuals for the above lifetime fits are shown below each measurement, and the y axis is in linear scale.

FIGURE 5.

LRET lifetimes for ΔNR1*T396C-Th, A715C:ΔNR2A* labeled with terbium chelate:ATTO 465 as measured by the sensitized emission of acceptor at 510 nm under saturating concentrations of agonists. The donor:acceptor lifetime shown here is the difference between the lifetimes obtained before and after thrombin digestion. The residuals are shown below the lifetime measurement, and the y axis is in linear scale.