Kinetic effects of perturbations at the NMDA receptor ligand -binding domains
Kussius, Cassandra Lynn
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NMDA receptor activity is required for synapse development, maintenance and plasticity; processes underlying the cellular mechanisms involved in learning and memory. Consequently, improper regulation of NMDA receptor activity has deleterious effects and is implicated in a host of neuropathologies. Physiologically, NMDA receptors respond to the neurotransmitter glutamate, and additionally require binding of the co-agonist glycine for activation. Functional receptors are obligate heterotetramers of two GluN1, glycine-binding, and two GluN2, glutamate-binding subunits. The agonist binding sites are located within a cleft between mobile lobes of extracellular ligand-binding domains in each subunit. Agonist binding is the first step in initiating a series of conformational changes that ultimately result in opening of the ion channel. In an attempt to increase our understanding of agonist induced activation, we investigated the kinetic effects of perturbations at the NMDA receptor ligand-binding domains. We selectively decreased and increased the stability of ligand-binding domain closure using partial agonists and engineered cross-cleft disulfide bonds, respectively. Single-channel recordings and kinetic analyses were used to measure the impact of these modifications on the NMDA receptor activation reaction. Partial agonists binding at either the glycine or the glutamate-site decreased the single-channel open probability through a combined effect of increased closed durations and decreased open durations. Regardless of the site of action, partial agonists worked through the same mechanism, suggesting that consecutive steps in the activation pathway are highly coupled. Stabilizing cleft-closure with disulfide bonds increased the single-channel open probability, but only when the GluN2A ligand-binding domain was locked closed. This indicates that glutamate does not fully stabilize this conformation on its own. Therefore, we propose that glutamate acts as a partial agonist at GluN2A containing NMDA receptors. These studies have increased our understanding of the NMDA receptor gating mechanism by identifying the transitions that are influenced by modifications at the ligandbinding domains. We found that alterations at the ligand-binding domains have wide spread effects on the activation pathway, serving to impede or enhance channel opening depending on whether the stability of cleft-closure was decreased or increased, respectively. The insight gained from these studies advances the goal of assigning structural identities to the conformational transitions involved in the NMDA receptor gating reaction. These studies will contribute to the overall effort to develop more effective pharmaceuticals to target the NMDA receptor in pathological conditions.