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. 2015 May 15;290(20):12812-20.
doi: 10.1074/jbc.M115.649723. Epub 2015 Mar 31.

Subtype-dependent N-methyl-D-aspartate Receptor Amino-Terminal Domain Conformations and Modulation by Spermine

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Free PMC article

Subtype-dependent N-methyl-D-aspartate Receptor Amino-Terminal Domain Conformations and Modulation by Spermine

Rita E Sirrieh et al. J Biol Chem. .
Free PMC article

Abstract

The N-methyl-d-aspartate (NMDA) subtype of the ionotropic glutamate receptors is the primary mediator of calcium-permeable excitatory neurotransmission in the central nervous system. Subunit composition and binding of allosteric modulators to the amino-terminal domain determine the open probability of the channel. By using luminescence resonance energy transfer with functional receptors expressed in CHO cells, we show that the cleft of the amino-terminal domain of the GluN2B subunit, which has a lower channel open probability, is on average more closed than the GluN2A subunit, which has a higher open probability. Furthermore, the GluN1 amino-terminal domain adopts a more open conformation when coassembled with GluN2A than with GluN2B. Binding of spermine, an allosteric potentiator, opens the amino-terminal domain cleft of both the GluN2B subunit and the adjacent GluN1 subunit. These studies provide direct structural evidence that the inherent conformations of the amino-terminal domains vary based on the subunit and match the reported open probabilities for the receptor.

Keywords: conformational change; fluorescence; fluorescence resonance energy transfer (FRET); glutamate receptor; ligand-binding protein.

Figures

FIGURE 1.
FIGURE 1.
ATD structure with suggested spermine-binding site and mutation sites for LRET experiments. A, crystal structure of the GluN1-GluN2B ATD dimer structure is shown highlighting the suggested spermine-binding residues in blue (PDB code 4PE5) (9, 10). B, schematics of individual subunits are shown indicating the locations of mutations and the resulting amino acid sequence. The Cys-free constructs involved the mutation to serine of Cys-22 and Cys-459, unless Cys-22 was retained to be labeled, and Cys-232, Cys-399, and Cys-495 in GluN2B, unless Cys-232 was retained to be labeled.
FIGURE 2.
FIGURE 2.
Different conformations adopted by apo-ATDs. A, LRET lifetimes of the apo-GluN2B cleft (black) and the Tricine-buffered GluN2A cleft (green) are shown. The GluN2A lifetime was originally published by Sirrieh et al. (23). B, LRET lifetimes from the GluN1 ATD vary when GluN1 is coexpressed with GluN2A (green) or GluN2B (black).
FIGURE 3.
FIGURE 3.
Spermine stabilizes an open cleft of both GluN1 and GluN2B ATDs. A, LRET measurements across the GluN2B clefts are shown. The measurements were made between His-30 and Cys-232 in GluN2B. B, LRET measurements between the upper and lower lobe of the GluN1 ATD. Measurements were made between Cys-22 and S224C in GluN1. C, donor-only measurements from the GluN2B subunit. D, donor-only measurements from the GluN1 subunit. In all panels, the black curve is from the apo-receptor, and the blue curve is from the spermine-bound receptor.
FIGURE 4.
FIGURE 4.
Electrophysiological characterization of the mutants used for the LRET experiments. A, sample trace is shown of spermine potentiating the whole cell currents obtained from NMDA receptors expressed in CHO cells. B, group data showing the extent of spermine potentiation for the various mutants as compared with the wild-type receptor. Error bars represent the S.E. All constructs, except for the wild type, are Cys-free, except for the indicated residues that were used for labeling in the spectroscopic measurements.
FIGURE 5.
FIGURE 5.
Spermine binding does not affect the upper lobes of the ATDs. A, these curves are the sensitized acceptor emission for receptors labeled at the upper lobes of the ATDs. The lifetime does not change when spermine is bound. B, donor-only lifetimes for the measurements between the upper lobes of the ATDs are shown. In all panels, the black curve is from the apo-receptor, and the blue curve is from the spermine-bound receptor.
FIGURE 6.
FIGURE 6.
Lower lobe of the GluN2B ATD rotates toward the upper lobe of the GluN1 ATD. A, LRET measurements between the lower lobe of the GluN2B ATD (Cys-232) and the upper lobe of the GluN1 ATD show that the lifetime decreases when spermine is bound. B, donor-only lifetimes for these constructs are shown. In all panels, the black curve is from the apo-receptor, and the blue curve is from the spermine-bound receptor.
FIGURE 7.
FIGURE 7.
Zinc induces no conformational change in the GluN1 ATD. A, LRET lifetimes of the GluN1 cleft when coexpressed with GluN2A do not change when the receptor is bound by zinc. The cyan curve is the apo-receptor, buffered by Tricine to remove contaminating zinc. Green shows the LRET decay from the zinc-bound receptor. B, donor-only lifetimes from the GluN1 cleft construct when coexpressed with GluN2A. The signal from the apo-receptor is in cyan and from the zinc-bound receptor is in cyan.
FIGURE 8.
FIGURE 8.
Working model. The GluN2A and GluN2B ATDs have inherently different conformations, which can be related to the open probability of the receptor. The GluN2A ATD adopts a more open conformation and has a correspondingly higher open probability, although the GluN2B ATD adopts a more closed conformation and has a lower open probability. Additionally, the GluN1 ATD adopts different conformations depending on the GluN2 subunit with which it is coexpressed. Spermine induces an opening of the GluN2B ATD.

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