Constitutive Activation of the N-Methyl-d-aspartate Receptor via Cleft-spanning Disulfide Bonds

Marie L Blanke,Antonius M.J Vandongen

doi:10.1074/jbc.m709190200

Abstract

Although the N-methyl-D-aspartate (NMDA) receptor plays a critical role in the central nervous system, many questions remain regarding the relationship between its structure and functional properties. In particular, the involvement of ligand-binding domain closure in determining agonist efficacy, which has been reported in other glutamate receptor subtypes, remains unresolved. To address this question, we designed dual cysteine point mutations spanning the NR1 and NR2 ligand-binding clefts, aiming to stabilize these domains in closed cleft conformations. Two mutants, E522C/I691C in NR1 (EI) and K487C/N687C in NR2 (KN) were found to exhibit significant glycine- and glutamate-independent activation, respectively, and co-expression of the two subunits produced a constitutively active channel. However, both individual mutants could be activated above constitutive levels in a concentration-dependent manner, indicating that cleft closure does not completely prevent agonist association. Interestingly, whereas the NR2 KN disulfide was found to potentiate channel gating and M3 accessibility, NR1 EI exhibited the opposite phenotype, suggesting that the EI disulfide may trap the NR1 ligand-binding domain in a lower efficacy conformation. Furthermore, both mutants affected agonist sensitivity at the opposing subunit, suggesting that closed cleft stabilization may contribute to coupling between the subunits. These results support a correlation between cleft stability and receptor activation, providing compelling evidence for the Venus flytrap mechanism of glutamate receptor domain closure.

Highlights

Strength, proposed to form the molecular basis of learning and memory [3]
Crystal structures obtained for the NR1 subunit in complex with full and partial agonists adopt a similar degree of domain closure; it appears that a different structural mechanism is required to account for partial agonist action at the NMDA receptor [15]
To further explore the activation mechanism of the NMDA receptor, we set out to investigate the role of cleft closure in sensing partial agonist efficacy, focusing on how cleft closure affects the accessibility of M3 and agonist sensitivity of both subunits

Summary

Introduction

Strength, proposed to form the molecular basis of learning and memory [3]. NMDA receptors have been implicated in the pathogenesis of numerous neurological disorders, including schizophrenia and Alzheimer disease, as well as neurodegeneration following ischemia or brain injury (4 – 6). Extensive studies with the GluR2 AMPA receptor, in complex with a range of full and partial agonists, established a correlation between agonist efficacy and degree of LBD closure, and a similar relationship was observed in kainate receptors [11, 13]. These results led to a structural model of receptor activation, in which agonist binding promotes LBD closure by rotating D2 toward D1, separating the linker regions and promoting channel opening. Partial agonist crystal structures have not yet been reported for NR2, so it remains to be determined whether the NR2 LBD behaves like NR1 or follows the AMPA/kainate receptor paradigm of partial agonist action

Methods

Results

Conclusion