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 alpha 2 delta-1 is a critical NMDAR-interacting protein and controls synaptic trafficking of NMDARs. In this study, we determined the relative roles of PKC and alpha 2 delta-1 in the control of NMDAR activity. We found that alpha 2 delta-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 alpha 2 delta-1. Remarkably, PKC inhibition with Go6983 abolished alpha 2 delta-1-coexpression-induced potentiation of NMDAR activity in cells transfected with GluN1/ GluN2A or GluN1/GluN2B. Treatment with PMA increased the alpha 2 delta-1-GluN1 interaction and promoted alpha 2 delta-1 and GluN1 cell surface trafficking. PMA also significantly increased NMDAR activity of spinal dorsal horn neurons and the amount of alpha 2 delta-1 bound GluN1 protein complexes in spinal cord synaptosomes in wild-type mice, but not in alpha 2 delta-1 knockout mice. Furthermore, inhibiting alpha 2 delta-1 with pregabalin or disrupting the alpha 2 delta-1-NMDAR interaction with the alpha 2 delta-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 alpha 2 delta-1. Together, our findings demonstrate the interdependence of alpha 2 delta-1 and PKC phosphorylation in regulating NMDAR trafficking and activity. The phosphorylation-dependent, dynamic alpha 2 delta-1-NMDAR interaction constitutes an important molecular mechanism of synaptic plasticity.