Single-Molecule FRET Investigations of Negative Cooperativity in the NMDA Receptor

Abstract

Ionotropic glutamate receptors mediate the majority of excitatory neurotransmission in the central nervous system. One subtype of ionotropic glutamate receptor, the N-methyl-D-aspartate receptor, is of note due to its requirement for not only glutamate, but also glycine, to bind to the receptor for activation to occur. The NMDA receptor achieves this co-agonist requirement due to its nature as an obligate heteromer consisting of two glycine-binding N1 subunits and two glutamate-binding N2 subunits, which are arranged in an alternating pattern. Glutamate and glycine exhibit negative cooperativity in the NMDA receptor, where the binding of one co-agonist causes a reduction in the affinity of the receptor for the other co-agonist. Herein we have utilized single-molecule fluorescence resonance energy transfer to study the negative cooperativity exhibited by the NMDA receptor. Our studies have shown that the glycine-binding domain in the apo state probes primarily an open conformation while occasionally also probing a closed conformation. The equilibrium between these open and closed conformations is shifted upon the binding of glycine, which indicates a conformational selection mechanism of receptor control. Furthermore, upon the binding of glutamate to the adjacent subunit, the glycine-binding domain will occasionally probe an open state similar to the one seen in the apo state. This shift in conformational stability would favor the unbinding of glycine from the receptor and could therefore be directly related to the negative cooperativity observed between the NMDA receptor co-agonists. In addition, the binding of glycine to the receptor causes the transmembrane segments to adopt a more tightly packed conformation than what is observed when the receptor is in the apo state. These results serve to reveal the conformational shifts that underlie various aspects of NMDA receptor behavior.