N-methyl D-aspartate receptors, are a subtype of glutamate receptors that mediate excitatory signal transmission in the mammalian central nervous system. Their primary function involves converting the chemical signal into an electrical signal, i.e. glutamate binding to an extracellular domain in the receptor triggers the formation of cation permeable transmembrane channels in the receptor. Given the importance of these receptors in mediating a number of physiological processes and the need to modulate their function in disease states, the primary questions are how does the agonist activate the protein and how can this mechanism be modulated. To address this question, we propose to use a combination of spectroscopic and biochemical methods to identify the complete conformational landscape of the agonist binding domain and the N-terminal modulator binding domain, in the presence of agonists and modulators that induce a large spectrum of activity. Our hypothesis based on the structures of the isolated components and the electrophysiological measurements of the NMDA receptor and the closely related AMPA receptors is that the activity of the receptor is controlled by the fraction of the agonist binding domain and the N-terminal domain that are in a closed cleft conformation in the receptor. We propose to test this hypothesis by using single molecule and ensemble FRET investigations to probe the conformational changes, which will then be correlated to functional consequences as determined by single channel and whole cell current recordings. Additionally, we hypothesize that the negative cooperativity between the agonist glycine and glutamate is mediated by the interface contacts within and across the dimers, in the dimer of dimer structure of the receptor. To test this hypothesis we will investigate the effect of stabilizing and destabilizing the dimer interface as well as the inter dimer interface in the agonist binding domain on the function as measured by the rates of agonist dissociation. The function based studies will be further corroborated structurally by measuring distance changes across the subunit using LRET. These functional and structural investigations will provide a comprehensive understanding of the mechanism by which agonists and modulators mediate NMDA receptor function. PUBLIC HEALTH RELEVANCE: NMDA receptors are a subtype of the glutamate receptor that mediate excitatory responses in the human central nervous system, and play an important role in controlling the cognitive and motor responses. Here we propose to determine the mechanistic and structural changes that underlie the activation and modulation of the function of the NMDA receptors. These investigations will provide insight at a molecular level as to how NMDA receptor function can be altered and hence aid in the treatment of the clinical conditions associated with it. .