Contribution of the M1 Transmembrane Helix and Pre-M1 Region to Positive Allosteric Modulation and Gating of N-Methyl-d-Aspartate Receptors

Abstract

N-methyl-D-aspartate (NMDA) receptors are glutamate-gated ion channels whose function is critical for normal excitatory synaptic transmission in the brain and whose dysfunction has been implicated in several neurologic conditions. NMDA receptor function is subject to extensive allosteric regulation both by endogenous compounds and by exogenous small molecules. Elucidating the structural determinants and mechanism of action by which allosteric regulators control gating will enhance our understanding of NMDA receptor activation and facilitate the development of novel therapeutics. Here, we investigated the structural determinants for (3-chlorophenyl)(6,7-dimethoxy-1-((4-methoxyphenoxy)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)methanone (CIQ), a GluN2C/ 2D-selective positive allosteric modulator. We show that CIQ does not bind to the amino-terminal domain of the NMDA receptor and does not share structural determinants with modulators acting at the agonist-binding domain dimer interface or ion channel pore. Rather, we identified critical determinants of CIQ modulation in the region near the first transmembrane helix of GluN2D, including in a putative pre-M1 cuff helix that may influence channel gating. We also show that mutations within the GluN2D pre-M1 region alter open probability of the NMDA receptor. These results suggest a novel site of action for potentiation of NMDA receptors by small molecules and implicate the pre-M1 region in NMDA receptor gating.

Molecular Mechanism of Disease-Associated Mutations in the Pre-M1 Helix of NMDA Receptors and Potential Rescue Pharmacology

“…Third, mutation of select pre-M1 residues to alanine can produce spontaneously active receptors [55]. Fourth, the pre-M1 helix and adjacent regions harbor structural determinants for subunit-selective positive allosteric modulators of the GluN2C- and GluN2D-containing NMDARs [56,57]. Fifth, mutations in and near pre-M1 can alter NMDAR function [14,29].…”

Allosteric modulators of NMDA receptors: multiple sites and mechanisms

“…Of those, only one residue (GluN2D-T592) differs between GluN2C/ GluN2D and GluN2A/2B subunits, which may thus participate in the subunit selectivity of CIQ. Mutations in the GluN2D pre-M1 helix, which runs parallel to the membrane and acts like a cuff around the ion channel gate region, also affect CIQ sensitivity [69]. Inspection of the location of the M1 and pre-M1 residues crucial for CIQ sensitivity according to the recent full-length NMDAR structures points to a binding site at an intersubunit cavity, which is partly lipid exposed and between two adjacent M1-M3 channel cores ( Figure 3).…”

“…Single-channel studies indicate that CIQ increases channel open probability by decreasing the mean shut time, suggesting that CIQ binding destabilizes closed states of the receptor channel [65 ]. CIQ exerts its effects likely through a novel binding site residing in the TMD [69]. A cluster of five residues in the N-terminal half of the first transmembrane helix (M1) of GluN2D appears crucial to potentiation by CIQ.…”

Two adjacent phenylalanines in the NMDA receptor GluN2A subunit M3 domain interactively regulate alcohol sensitivity and ion channel gating

“…For GluN2A F636 and F637, however, any functional interaction between the positions would be dependent upon additional bonds of one or both positions with nearby residues on other parts of the protein, as discussed above. The presence of these additional bonds appears likely, because positions in the M3 domain have been shown to interact with multiple positions in the M1 and M2 domains in non-NMDA glutamate receptors and NMDA receptors (Ogden and Traynelis, 2013; Lopez et al , 2013; Siegler Retchless et al , 2012; Xu et al , 2015; Wilding et al , 2014). We envision the interactions involving tryptophan as “balancing” this part of the M3 helix, such that the interactions of 636 and 637 with other side chains to alter channel closing rate effectively offset each other (Fig.…”