Mechanism of Activation in NMDA Receptors

Abstract

The N-methyl D-aspartate receptors belong to the family of ionotropic glutamate receptors. They are obligate heteromeric receptors with two glycine binding subunits (GluN1) and two glutamate binding (GluN2A-D) subunits arranged as a dimer of dimers. We have used luminescence resonance energy transfer measurements to determine the specific arrangement of the GluN1 and GluN2 subunits within the tetramer. Based on this arrangement we have designed sites in the agonist binding domain that can probe changes in distances within the subunit and not across the subunits. By tagging these sites with donor and acceptor fluorophores we have investigated the conformational changes in the agonist binding domain of the GluN1 and GluN2A subunits using luminescence resonance energy transfer. The LRET based distances indicate a cleft closure conformational change at the GluN1 subunit upon binding agonists, however, no significant changes in the cleft closure are observed between partial and full agonists. Single molecule FRET investigations are being performed to determine if the partial agonist bound state exhibits more flexibility relative to the full agonist bound state, accounting for the lower activation. The LRET-based distances for the glutamate binding GluN2A subunit, on the other hand, shows a graded cleft closure upon binding agonists with varying efficacy, with the extent of cleft closure being proportional to the extent of activation. This graded cleft closure indicates that the mechanism of activation in this subunit is similar to that of the glutamate binding α-amino-5-methyl-3-hydroxy-4-isoxazole propionate and kainate subtypes of the ionotropic glutamate receptors.