Complex functional phenotypes of NMDA receptor disease variants

NMDA receptors have essential roles in the physiology of central excitatory synapses and their dysfunction causes severe neuropsychiatric symptoms. Recently, a series of genetic variants have been identified in patients, however, functional information about these variants is sparse and their role in pathogenesis insufficiently known. Here we investigate the mechanism by which two GluN2A variants may be pathogenic. We use molecular dynamics simulation and single-molecule electrophysiology to examine the contribution of GluN2A subunit-residues, P552 and F652, and their pathogenic substitutions, P552R and F652V, affect receptor functions. We found that P552 and F652 interact during the receptors normal activity cycle; the interaction stabilizes receptors in open conformations and is required for a normal electrical response. Engineering shorter side-chains at these positions (P552A and/or F652V) caused a loss of interaction energy and produced receptors with severe gating, conductance, and permeability deficits. In contrast, the P552R sidechain resulted in stronger interaction and produced a distinct, yet still drastically abnormal electrical response. These results identify the dynamic contact between P552 and F652 as a critical step in the NMDA receptor activation, and show that both increased and reduced communication through this interaction cause dysfunction. Results show that subtle differences in NMDA receptor primary structure can generate complex phenotypic alterations whose binary classification is too simplistic to serve as a therapeutic guide. Main findingsO_LITwo NMDA receptor residues whose substitution results in encephalopathies, were found to form new interactions during activation, and the energy provided by this interaction is required for normal receptor gating. C_LIO_LIExperimental substitutions of these residues that change the strength of their interaction reduce the receptor open probability, unitary conductance, and calcium permeability. C_LIO_LIReceptors with variations at these positions identified in patients display a broad range of both gain- and loss-of-function changes depending on the stimulation protocol. C_LI