Single Molecule FRET Investigations of the NMDA Receptor

Abstract

Glutamate receptors are responsible for the majority of excitatory signaling in the central nervous system. These receptors, of which the NMDA receptor is one subtype, mediate communication between neurons by converting chemical signals in the form of neurotransmitters to electrical signals in the form of ion flow through transmembrane channels. NMDA receptors accomplish this signal conversion by binding to two co-agonist neurotransmitters, glycine and glutamate, which results in conformational changes that ultimately lead to the opening of a cation-permeable pore in the plasma membrane. It has been observed previously that the two co-agonists glycine and glutamate exhibit negative cooperativity, that is, the affinity of one agonist is reduced by the presence of the other agonist. The mechanism of this negative cooperativity, however, has not been elucidated. We have utilized single molecule FRET measurements, as well as molecular dynamics simulations and electrophysiological measurements, to examine the mechanism of negative cooperativity in the NMDA receptor. We have found that the binding of one agonist leads to a conformational change in the binding site for the other agonist that disfavors agonist binding and causes an increase in conformational dynamics. The loss of this conformational change in a mutant receptor that does not exhibit negative cooperativity confirms the involvement of the identified conformational states in mediating the negative cooperativity. Additionally, measurements at the transmembrane region of the receptor indicate that the binding of both agonists is required to induce the opening of the cation pore. These results demonstrate the importance of using single molecule measurements to elucidate all of the conformational states that a protein probes and also serve to increase our understanding of the mechanisms underlying agonist affinity in the NMDA receptor.