Protein Kinase C-Mediated Phosphorylation and α2δ-1 Interdependently Regulate NMDA Receptor Trafficking and Activity

N-methyl-d-aspartate receptors (NMDARs) are important for synaptic plasticity associated with many physiological functions and neurologic disorders. Protein kinase C (PKC) activation increases the phosphorylation and activity of NMDARs, and α2δ-1 is a critical NMDAR-interacting protein and controls synaptic trafficking of NMDARs. In this study, we determined the relative roles of PKC and α2δ-1 in the control of NMDAR activity. We found that α2δ-1 coexpression significantly increased NMDAR activity in HEK293 cells transfected with GluN1/GluN2A or GluN1/GluN2B. PKC activation with phorbol 12-myristate 13-acetate (PMA) increased receptor activity only in cells coexpressing GluN1/GluN2A and α2δ-1. Remarkably, PKC inhibition with Gӧ6983 abolished α2δ-1-coexpression-induced potentiation of NMDAR activity in cells transfected with GluN1/GluN2A or GluN1/GluN2B. Treatment with PMA increased the α2δ-1-GluN1 interaction and promoted α2δ-1 and GluN1 cell surface trafficking. PMA also significantly increased NMDAR activity of spinal dorsal horn neurons and the amount of α2δ-1-bound GluN1 protein complexes in spinal cord synaptosomes in wild-type mice, but not in α2δ-1 knockout mice. Furthermore, inhibiting α2δ-1 with pregabalin or disrupting the α2δ-1-NMDAR interaction with the α2δ-1 C-terminus peptide abolished the potentiating effect of PMA on NMDAR activity. Additionally, using quantitative phosphoproteomics and mutagenesis analyses, we identified S929 on GluN2A and S1413 (S1415 in humans) on GluN2B as the phosphorylation sites responsible for NMDAR potentiation by PKC and α2δ-1. Together, our findings demonstrate the interdependence of α2δ-1 and PKC phosphorylation in regulating NMDAR trafficking and activity. The phosphorylation-dependent, dynamic α2δ-1-NMDAR interaction constitutes an important molecular mechanism of synaptic plasticity.SIGNIFICANCE STATEMENT A major challenge in studies of protein phosphorylation is to define the functional significance of each phosphorylation event and determine how various signaling pathways are coordinated in response to neuronal activity to shape synaptic plasticity. PKC phosphorylates transporters, ion channels, and G-protein-coupled receptors in signal transduction. In this study, we showed that α2δ-1 is indispensable for PKC-activation-induced surface and synaptic trafficking of NMDARs, whereas the α2δ-1-NMDAR interaction is controlled by PKC-induced phosphorylation. Our findings reveal that α2δ-1 mainly functions as a phospho-binding protein in the control of NMDAR trafficking and activity. This information provides new mechanistic insight into the reciprocal roles of PKC-mediated phosphorylation and α2δ-1 in regulating NMDARs and in the therapeutic actions of gabapentinoids.