Subunit Arrangement and Phenylethanolamine Binding in GluN1/GluN2B NMDA Receptors

Summary

Since it was unexpectedly discovered that the anti-hypertensive agent, ifenprodil, has neuroprotective activity through effects to N-methyl-D-aspartate (NMDA) receptors ¹ , enormous efforts have been made to understand the mechanism of action and to develop improved therapeutic compounds based on this knowledge ^{2– 4} . Neurotransmission mediated by NMDA receptors is essential for basic brain development and function ⁵ . These receptors form heteromeric ion channels and become activated upon 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/GluN2B NMDA receptors ^{6, 7} , have been intensely studied for their potential use in treatment of various neurological disorders and diseases including depression, Alzheimer’s disease and Parkinson’s disease ^{2, 4} . Despite great enthusiasm, mechanisms underlying recognition of phenylethanolamines and the ATD-mediated allosteric inhibition remain limited due to lack of structural information. Here we report that the GluN1 and GluN2B ATDs form heterodimer and that phenylethanolamine binds at the GluN1-GluN2B subunit interface rather than within the GluN2B cleft. The crystal structure of the GluN1b/GluN2B ATD heterodimer shows a highly distinct pattern of subunit arrangement that is different from those observed in homodimeric non-NMDA receptors and reveals the molecular determinants for phenylethanolamine binding. Restriction of domain movement in the bi-lobed structures of the GluN2B ATD by engineering an inter-subunit disulfide bond dramatically decreases ifenprodil-sensitivity indicating that conformational freedom in the GluN2B ATD is essential for ifenprodil-mediated allosteric inhibition in NMDA receptors.