Project Summary N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels composed of transmembrane GluN1, GluN2 (A-D), and GluN3 (A-B) subunits that mediate Ca2+ influx into the dendritic spine. Importantly, the unique intracellular tail of the GluN2B subunit is essential for learning and memory. Furthermore, autism spectrum disorder (ASD) and schizophrenia (SCZ) associated variants are found within the GluN2B intracellular tail. My overall goal is to elucidate a novel mechanism by which GluN2B tails contribute to the function of GluN2B- containing NMDARs (GluN2B-NMDARs). Previous experiments showed that actin-targeting drugs impact NMDAR activity, but the underlying mechanisms are unknown. My preliminary data strongly support the primary hypothesis of this project: a direct interaction between actin filaments and the GluN2B intracellular tail regulates NMDAR activity. The objective of my proposed project is to elucidate the biochemical basis for an interaction between the GluN2B tail and actin filaments and determining how this interaction regulates NMDAR function. I will achieve this objective by pursuing three highly complementary aims: Aim 1. Define the minimal fragment of GluN2B intracellular tail that mediates high affinity binding to actin filaments and the interfaces on GluN2B and actin that mediate binding. My preliminary data show a direct interaction between GluN2B tails and actin, but the key interfaces that mediate this interaction between the proteins is unknown. I will use recombinant proteins to perform quantitative binding assays and cross-linking assays to identify the minimal fragment of the GluN2B tail needed to mediate a high affinity interaction with actin. Aim 2. Characterize how Ca2+ and genetic variants impact GluN2B:actin binding. Ca2+ decreases NMDAR activity in cultured hippocampal neurons, but whether Ca2+ selectively impacts GluN2B-NMDAR activity is unknown. My preliminary data suggest Ca2+ strengthens the GluN2B tail:actin interaction. Genetic variants that lie within GluN2B tail regions that I found bind actin have been identified in patients with ASD and SCZ. Clusters of conserved and positively charged residues can contribute to actin binding of proteins. I will perform quantitative binding assays to identify how Ca2+ levels, disease-associated variants, and clusters of conserved and positive residues impact the GluN2B tail:actin interaction. Aim 3: Determine how manipulations of the actin cytoskeleton and GluN2B tail impact GluN2B-NMDAR activity. Changes in actin polymer state impact NMDAR activity in neurons. However, whether actin selectively impacts GluN2B-NMDARs or is mediated by GluN2B tail:actin interactions is unclear. I will monitor changes in GluN2B-NMDAR mediated currents in HEK293 cells to determine the impact of 1) actin-targeting drugs and 2) disrupting the GluN2B tail:actin interaction. After mastering these techniques, I will monitor how actin dynamics and GluN2B tail:actin interac...