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Subunit arrangement and phenylethanolamine binding in GluN1/GluN2B NMDA receptors


Since it was discovered that the anti-hypertensive agent ifenprodil has neuroprotective activity through its effects on NMDA (N-methyl-D-aspartate) receptors1, a determined effort has been made to understand the mechanism of action and to develop improved therapeutic compounds on the basis of this knowledge2,3,4. Neurotransmission mediated by NMDA receptors is essential for basic brain development and function5. These receptors form heteromeric ion channels and become activated after concurrent binding of glycine and glutamate to the GluN1 and GluN2 subunits, respectively. A functional hallmark of NMDA receptors is that their ion-channel activity is allosterically regulated by binding of small compounds to the amino-terminal domain (ATD) in a subtype-specific manner. Ifenprodil and related phenylethanolamine compounds, which specifically inhibit GluN1 and GluN2B NMDA receptors6,7, have been intensely studied for their potential use in the treatment of various neurological disorders and diseases, including depression, Alzheimer’s disease and Parkinson’s disease2,4. Despite considerable enthusiasm, mechanisms underlying the recognition of phenylethanolamines and ATD-mediated allosteric inhibition remain limited owing to a lack of structural information. Here we report that the GluN1 and GluN2B ATDs form a heterodimer and that phenylethanolamine binds at the interface between GluN1 and GluN2B, rather than within the GluN2B cleft. The crystal structure of the heterodimer formed between the GluN1b ATD from Xenopus laevis and the GluN2B ATD from Rattus norvegicus shows a highly distinct pattern of subunit arrangement that is different from the arrangements observed in homodimeric non-NMDA receptors and reveals the molecular determinants for phenylethanolamine binding. Restriction of domain movement in the bi-lobed structure of the GluN2B ATD, by engineering of an inter-subunit disulphide bond, markedly decreases sensitivity to ifenprodil, indicating that conformational freedom in the GluN2B ATD is essential for ifenprodil-mediated allosteric inhibition of NMDA receptors. These findings pave the way for improving the design of subtype-specific compounds with therapeutic value for neurological disorders and diseases.

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Figure 1: Binding of phenylethanolamine requires both GluN1b and GluN2B ATDs, and stabilizes heterodimers.
Figure 2: Structure of the GluN1b–GluN2B ATD heterodimer in complex with ifenprodil at 2.6 Å resolution.
Figure 3: Phenylethanolamine binding site.
Figure 4: Engineering of disulphide bonds at the subunit interface alters sensitivity to ifenprodil.

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Protein Data Bank

Data deposits

Structural coordinates are deposited in the Protein Data Bank with accession codes 3QEK for GluN1b ATD, 3QEL for GluN1b–GluN2B ATD in complex with ifenprodil and 3QEM for GluN1b–GluN2B ATD in complex with Ro 25-6981.


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We thank the staff at X25 and X29 at the National Synchrotron Light Source for beamline support. M. Mayer is thanked for comments on this work. We also thank D. Raleigh for the use of analytical ultracentrifugation. GluN1 clones from Xenopus laevis were gifts from M. Hollmann and H. Cline. This work was supported by NIH MH085926, the Alzheimer’s Association and a donation from the Fox family (to H.F.). H.F. was also funded by a scientist development grant from the American Heart Association. E.K. is supported by a NARSAD Lieber Young Investigator Award.

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The project was initiated by E.K. and H.F. All of the experiments were designed by E.K. and H.F. Crystallographic studies, isothermal calorimetry and analytical ultracentrifugation were carried out by E.K. Electrophysiology and crosslinking experiments were conducted by H.F. Technical support was given by N.S. The manuscript was written by H.F. and E.K.

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Correspondence to Hiro Furukawa.

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The authors declare no competing financial interests.

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Karakas, E., Simorowski, N. & Furukawa, H. Subunit arrangement and phenylethanolamine binding in GluN1/GluN2B NMDA receptors. Nature 475, 249–253 (2011).

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