InCA1 pyramidal neurons of the hippocampus protein kinase C regulates calcium-dependent inactivation of NMDA receptors

The NMDA subtype of the glutamate-gated channel exhibits a high permeability to Ca2+. The influx of Ca2+ through NMDA channels is limited by a rapid and Ca2+/calmodulin (CaM)-dependent inactivation that results from a competitive displacement of cytoskeleton-binding proteins from the NR1 subunit of the receptor by Ca2+/CaM (Zhang et al., 1998; Krupp et al., 1999). The C terminal of this subunit can be phosphorylated by protein kinase C (PKC) (Tingley et al., 1993). The present study sought to investigate whether PKC regulates Ca2+ dependent inactivation of the NMDA channel in hippocampal neurons. Activation of endogenous PKC by 4 beta-phorbol 12-myristate 13-acetate enhanced peak (Ip) and depressed steady-state (I-ss) NMDA-evoked currents, resulting in a reduction in the ratio of these currents (I-ss/I-p). We demonstrated previously that PKC activity enhances I-P via a sequential activation of the focal adhesion kinase cell adhesion kinase beta/proline-rich tyrosine kinase 2 (CAK beta/Pyk2) and the nonreceptor tyrosine kinase Src (Huang et al., 1999; Lu et al., 1999). Here, we report that the PKC-induced depression of Iss is unrelated to the PKC/CAK beta/Src-signaling pathway but depends on the concentration of extracellular Ca2+. Intracellular applications of CaM reduced I-ss/I-p and occluded the Ca2+ dependent effect of phorbol esters on I-ss. Moreover, increasing the concentration of intracellular Ca2+ buffer or intracellular application of the inhibitory CaM-binding peptide (KY9) greatly reduced the phorbol ester-induced depression of I-ss. Taken together, these results suggest that PKC enhances Ca2+/CaM-dependent inactivation of the NMDA channel, most likely because of a phosphorylation-dependent regulation of interactions between receptor subunits, CaM, and other postsynaptic density proteins.