Inter-Subunit Interactions of NMDA Receptor Amino-Terminal Domains Associated with Allosteric Modulation

Abstract

N-methyl-D-aspartate (NMDA) receptors are one of the three main types of ionotropic glutamate receptors. NMDA receptors are obligate heterotetramers typically composed of two GluN1 and two GluN2 subunits. GluN1 subunits can be one of eight different splice variants, and GluN2 subunits are classified as four subtypes A-D. Each subunit is organized into domains: an intracellular carboxy-terminal domain, the transmembrane, pore-forming domain, an extracellular, ligand-binding domain, and an extracellular, amino-terminal domain (ATD). NMDA receptors are unique among glutamate receptors because allosteric modulators bind to the ATD and either inhibit or potentiate the receptor. The unique feature of these modulators is the subunit specific nature of their effect, despite relatively high sequence homology (∼56% for GluN2A and GluN2B ATDs). For instance, zinc inhibits NMDA receptors containing different GluN2 subunits with varying affinities and efficacies. Ifenprodil only inhibits receptors containing the GluN2B subtype, and spermine only potentiates receptors containing the GluN2B subtype. Luminescence resonance energy transfer (LRET) on full-length receptors expressed in CHO cells was used to characterize conformational changes associated with the binding of allosteric modulators to the amino-terminal domains. Zinc binding induces a cleft-closure conformational change in the GluN2A ATD, and the lower lobe of the GluN2A ATD moves towards the upper lobe of the GluN1 ATD. There is no change in the distance between the two upper lobes of the GluN1 and GluN2A ATDs. Finally, LRET measurements show that zinc binding to the GluN2A ATD does not allosterically induce any large scale movements within the GluN1 ATD. These findings suggest that the cleft-closure conformational change in the GluN2A ATD upon binding zinc is most likely only propagated down towards the ligand binding domain and ultimately the channel segments.