Mechanistic Basis of NMDA Receptor Channel Property Variation

Glutamate mediates the majority of fast excitatory neurotransmission in the vertebrate brain. Glutamate receptors (GluRs) transduce signals in two ways: metabotropic GluRs signal via intracellular G proteins, whereas ionotropic GluRs (iGluRs) open intrinsic ion channels in response to agonist binding. NMDA receptors (NMDARs) are glutamate- and glycine-gated iGluRs that play critical roles in spatial learning, contextual fear memory acquisition, synapse elimination, and chronic pain. Their particularly high calcium (Ca2+) permeability and strongly voltage-dependent channel block by external magnesium (Mg2+) distinguish NMDARs from other iGluRs. Mg2+ channel block of NMDARs inhibits current influx through the majority of agonist-bound, open NMDARs at resting membrane potentials (Vms), but this block is relieved by depolarization. Thus, significant current flow through NMDARs requires presynaptic activity (glutamate release) and postsynaptic activity (depolarization to relieve Mg2+ channel block), conferring on NMDARs a coincidence-detection capability that is central to their physiological importance. To mediate this and other important functions, NMDARs require tight regulation of the voltage-dependent Mg2+ block that provides crucial control of NMDAR-mediated current flow and Ca2+ influx. NMDARs are typically composed of NR1 and NR2 subunits. The four NR2 subunits (NR2A-D) contribute to four diheteromeric NMDAR subtypes (NR1/2A-NR1/2D), which differ in many respects, including the magnitudes of channel block by Mg2+, Ca2+ permeability, and single-channel conductance. Previously-gathered data from our lab demonstrates that the subtype specificity of Mg2+ block is principally conferred by a single amino acid site in the third transmembrane region (M3) of NR2 subunits. This "NR2 S/L site" contains a serine in NR2A and NR2B subunits and a leucine in NR2C and NR2D subunits. Surprisingly, the NR2 S/L site does not line the pore. I created several structural homology models of NMDARs to generate hypotheses regarding how the NR2 S/L site conveys its effects to the pore. I tested these hypotheses experimentally and found that the NR2 S/L site interacts with an NR1 subunit tryptophan in the pore-loop to regulate Mg2+ block properties. I further determined that the NR2 S/L site greatly contributes to the subtype variation in single-channel conductance, and likely plays a role in the subtype variation in Ca2+ permeability

Q/R site interactions with the M3 helix in GluK2 kainate receptor channels revealed by thermodynamic mutant cycles

RNA editing at the Q/R site near the apex of the pore loop of AMPA and kainate receptors controls a diverse array of channel properties, including ion selectivity and unitary conductance and susceptibility to inhibition by polyamines and cis-unsaturated fatty acids, as well as subunit assembly into tetramers and regulation by auxiliary subunits. How these different aspects of channel function are all determined by a single amino acid substitution remains poorly understood; however, several lines of evidence suggest that interaction between the pore helix (M2) and adjacent segments of the transmembrane inner (M3) and outer (M1) helices may be involved. In the present study, we have used double mutant cycle analysis to test for energetic coupling between the Q/R site residue and amino acid side chains along the M3 helix. Our results demonstrate interaction with several M3 locations and particularly strong coupling to substitution for L614 at the level of the central cavity. In this location, replacement with smaller side chains completely and selectively reverses the effect of fatty acids on gating of edited channels, converting strong inhibition of wild-type GluK2(R) to nearly 10-fold potentiation of GluK2(R) L614A

Influence of GluN2 subunit identity on NMDA receptor function

AbstractN-methyl-d-aspartate receptors (NMDARs) are ligand-gated ion channels (‘ionotropic’ receptors) activated by the major excitatory neurotransmitter, l-glutamate. While the term ‘the NMDAR’ is often used it obscures the fact that this class of receptor contains within it members whose properties are as different as they are similar. This heterogeneity was evident from early electrophysiological, pharmacological and biochemical assessments of the functional properties of NMDARs and while the molecular basis of this heterogeneity has taken many years to elucidate, it indicated from the outset that the diversity of NMDAR phenotypes could allow this receptor family to subserve a variety of functions in the mammalian central nervous system. In this review we highlight some recent studies that have identified structural elements within GluN2 subunits that contribute to the heterogeneous biophysical properties of NMDARs, consider why some recently described novel pharmacological tools may permit better identification of native NMDAR subtypes, examine the evidence that NMDAR subtypes differentially contribute to the induction of long-term potentiation and long-term depression and discuss how through the use of chimeric proteins additional insights have been obtained that account for NMDAR subtype-dependency of physiological and pathophysiological signalling.This article is part of the Special Issue entitled ‘Glutamate Receptor-Dependent Synaptic Plasticity’

Mechanism of differential control of NMDA receptor activity by NR2 subunits

International audienceNMDA receptors (NMDARs) are a major class of excitatory neurotransmitter receptors in the central nervous system. They form glutamate-gated ion channels highly permeable to calcium that mediate activity-dependent synaptic plasticity1. NMDAR dysfunction is implicated in multiple brain disorders, including stroke, chronic pain and schizophrenia2. NMDARs exist as multiple subtypes with distinct pharmacological and biophysical properties largely determined by the type of NR2 subunit (NR2A-NR2D) incorporated in the heteromeric NR1/NR2 complex1,3,4. A fundamental difference between NMDAR subtypes is their channel maximal open probability (Po), which spans a 50-fold range from ~0.5 for NR2A-containing receptors to ~0.01 for NR2C-and NR2D-containing receptors; NR2B-containing receptors having an intermediate value (~0.1)5–9. These differences in Po confer unique charge transfer capacities and signaling properties on each receptor subtype4,6,10,11. The molecular basis for this profound difference in activity between NMDAR subtypes is unknown. Here we demonstrate that the subunit-specific gating of NMDARs is controlled by the region formed by the NR2 N-terminal domain (NTD), an extracellular clamshell-like domain previously shown to bind allosteric inhibitors12–15, and the short linker connecting the NTD to the agonist-binding domain (ABD). Subtype specificity of NMDAR Po largely reflects differences in the spontaneous (ligand-independent) equilibrium between open-cleft and closed-cleft conformations of the NR2-NTD. This NTD-driven gating control also impacts pharmacological properties, by setting the sensitivity to the endogenous inhibitors zinc and protons. Our results provide a proof-of-concept for a drug-based bidirectional control of NMDAR activity using molecules acting either as NR2-NTD " closers " or " openers " promoting receptor inhibition or potentiation, respectively. Keyword