Subunit-Specific Activation of NMDA Receptors

Abstract

NMDA receptors are ligand-gated ion channels assembled from two NR1 and two NR2 subunits, and are activated upon simultaneous binding of glycine and glutamate to the NR1 and NR2 subunits, respectively. The different NR2 subunits (NR2A-D) endow the NMDA receptors with markedly different biophysical and pharmacological properties. We have focused on how the conformational changes that are induced by agonist binding and enable channel gating are specific for the NR2 subunit. For this purpose, we have developed a series of N-hydroxypyrazole-5-glycine (NHP5G) compounds that are partial agonists for the glutamate binding site of the NR2 subunit. This structurally related series of partial agonists show a broad range of relative efficacies at NR2 subunits and weakly activate the channel, allowing better identification of the steps associated with channel opening. Propyl-substituted NHP5G shows strong subunit selectivity in that it activates NR1/NR2D (37%), but does not appear to activate NR1/NR2A (∼0%).Single-channel recordings for NR1/NR2D expressed in HEK293 cells using partial agonists (NH5PG, ethyl-, and propyl-NH5PG) and the full agonist glutamate enable us to determine which states in the process of channel activation can be modulated in an agonist-specific manner. These data show that glutamate, NHP5G, ethyl-, and propyl-NH5PG have strikingly different mean open times for NR1/NR2D (0.747, 0.432, 0.319, and 0.181 ms, respectively). We are currently comparing the rate constants for activation in models fitted to data from different partial agonists. We have also performed molecular dynamics (MD) simulations of the agonist binding domains for NR1/NR2A and NR1/NR2D bound with glutamate or propyl-NHP5G to predict how the agonists interact differentially with receptor subtypes. The synthesis of these lines of investigation will be used to identify structural elements that can be modified using mutagenesis to test working hypotheses on the structural basis for subunit-specific activation of NMDA receptors.