Positive Modulatory Interactions of NMDA Receptor GluN1/2B Ligand Binding Domains Attenuate Antagonists Activity

Abstract

N-methyl D-aspartate receptors (NMDAR) play crucial role in normal brain function and pathogenesis of neurodegenerative and psychiatric disorders. Functional tetra-heteromeric NMDAR contains two obligatory GluN1 subunits and two identical or different non-GluN1 subunits that include six different gene products; four GluN2 (A-D) and two GluN3 (A-B) subunits. The heterogeneity of subunit combination facilities the distinct function of NMDARs. All GluN subunits contain an extracellular N-terminal Domain (NTD) and ligand binding domain (LBD), transmembrane domain (TMD) and an intracellular C-terminal domain (CTD). Interaction between the GluN1 and co-assembling GluN2/3 subunits through the LBD has been proven crucial for defining receptor deactivation mechanisms that are unique for each combination of NMDAR. Modulating the LBD interactions has great therapeutic potential. In the present work, by amino acid point mutations and electrophysiology techniques, we have studied the role of LBD interactions in determining the effect of well-characterized pharmacological agents including agonists, competitive antagonists, and allosteric modulators. The results reveal that agonists (glycine and glutamate) potency was altered based on mutant amino acid sidechain chemistry and/or mutation site. Most antagonists inhibited mutant receptors with higher potency; interestingly, clinically used NMDAR channel blocker memantine was about three-fold more potent on mutated receptors (N521A, N521D, and K531A) than wild type receptors. These results provide novel insights on the clinical pharmacology of memantine, which is used for the treatment of mild to moderate Alzheimer's disease. In addition, these findings demonstrate the central role of LBD interactions that can be exploited to develop novel NMDAR based therapeutics.

Keywords: Ligand Binding Domain (LBD); NMDA receptor; competitive antagonists; interface; memantine.

Figure 1

Effect of GluN1 LBD mutations (co-expressed with GluN2B) on the activity of LBD cleft binding compounds. (A) topology diagram shows the location of different domains (NTD, LBD, and TMD) in the GluN1 and GluN2B dimer. Glycine (orange circle) and glutamate (brown circle), ifenprodil (triangle), and memantine (inverted triangle) binding is marked. LBD interaction sites are labeled as 1 (Site-I, blue), 2 (Site-II, red), and 3 (Site-III, green). Following are the mutations made at these sites: N521A, N521D at site-I; K531A & Y535A at Site-II and E781A at Site-III. Mutation induced changes in potency of the co-agonist, glycine (Bi,ii) and agonist glutamate (Ci,ii) of the NMDA receptor. Traces represent dose response curves (black-wildtype, red-Y535A, green-E781A; gray and brown bar- agonist and antagonist, respectively) that show results obtained from glycine site antagonist, 5,7-DCKA (Di–iii, scale: X-axis 10 s, and Y-axis 25 nA) and the effect of glutamate site antagonist, DL-AP5 (Ei–iii, scale: X-axis 10 s, and Y-axis 100 nA). Statistical significance is marked as ^*p < 0.01 or ^**P < 0.001.

Figure 2

Effect of GluN1 LBD mutations expressed with GluN2B on the activity of non-competitive antagonists. Dose response curves and histograms show changes in potency of ifenprodil (Ai,ii), and channel blockers Zn²⁺ (Bi,ii) and memantine (Ci,ii); C.iii shows the % reduction in memantine efficacy with Y535A mutant. Statistical significance is marked as ^*p < 0.01 or ^**P < 0.001.