LAUSR

N‐methyl‐d‐aspartate receptors (NMDARs) are calcium‐permeable ion channels that are ubiquitously expressed within the glutamatergic postsynaptic density. Phosphorylation of NMDAR subunits defines receptor conductance and surface localization, two alterations that can modulate overall channel activity. Modulation of NMDAR phosphorylation by kinases and phosphatases regulates the amount of calcium entering the cell and subsequent activation of calcium‐dependent processes. The dendritic spine enriched protein, spinophilin, is the major synaptic protein phosphatase 1 (PP1) targeting protein. Depending on the substrate, spinophilin can act as either a PP1 targeting protein, to permit substrate dephosphorylation, or a PP1 inhibitory protein, to enhance substrate phosphorylation. Spinophilin limits NMDAR function in a PP1‐dependent manner. Specifically, we have previously shown that spinophilin sequesters PP1 away from the GluN2B subunit of the NMDAR, which results in increased phosphorylation of Ser‐1284 on GluN2B. However, how spinophilin modifies NMDAR function is unclear. Herein, we utilize a Neuro2A cell line to detail that Ser‐1284 phosphorylation increases calcium influx via GluN2B‐containing NMDARs. Moreover, overexpression of spinophilin decreases GluN2B‐containing NMDAR activity by decreasing its surface expression, an effect that is independent of Ser‐1284 phosphorylation. In hippocampal neurons isolated from spinophilin knockout animals, there is an increase in cleaved caspase‐3 levels, a marker of calcium‐associated apoptosis, compared with wildtype mice. Taken together, our data demonstrate that spinophilin regulates GluN2B containing NMDAR phosphorylation, channel function, and trafficking and that loss of spinophilin enhances neuronal cleaved caspase‐3 expression.