Mapping Structural Elements to NMDA Receptor Activation Steps

Abstract

NMDA receptors are heterotetrameric glutamate-activated ionotropic receptors that mediate excitatory synaptic transmission in the brain. Numerous NMDA receptor subunit mutations are associated with epilepsy and several have been identified as causal factors. Single-molecule electrophysiological recordings reveal that NMDA receptors explore many functional configurations. However, our ability to map structural changes to each of these functional configurations has been limited. Here, we integrate single-molecule recordings of GluN1-1a/GluN2A receptors with computational modeling of receptor activation to determine structural elements necessary for receptor activation. We found using single-channel recordings of disease-associated mutation, GluN1-1a I642L (GluN1-1aI642L) reduced receptor open probability within bursts (wild-type: 0.72 ± 0.04; GluN1-1aI642L: 0.04 ± 0.06), mean open duration (wild-type: 4.34 ± 0.11 msec; GluN1-1aI642L: 1.10 ± 0.09 msec), and mean closed duration within bursts (wild-type: 3.21 ± 0.51 msec; GluN1-1aI642L: 11.74 ± 1.20 msec). To determine the structural mechanism by which this disease mutation perturbs channel function, we used GluN1/GluN2B crystal structures to build a homology model of GluN1/GluN2A lacking cytoplasmic and amino terminal domains. Using this model, we performed targeted molecular dynamics simulations of the closed receptor into a putative “open” conformation. We found that residue GluN1 I642 forms additional atomic contacts with L550 on the neighboring GluN2A during this transition. We performed mutant cycle analysis on these pair of residues by recording single-channel currents through GluN1-1a/GluN2A, GluN1-1AI642L/GluN2A, GluN1-1a/GluN2AL550I, or GluN1-1AI642L/GluN2AL550I. Using kinetic modeling of these currents to measure the free energy change associated with each transition step in the receptor activation pathway, we found that the strongest coupling between GluN1 I642 and GluN2A L550 occurs transitions between states C3 to C2 and O1 to O2. Our approach provides a tool by which we can begin to map precise structural elements necessary for each transition step in receptor function.