Abstract

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 4β-phorbol 12-myristate 13-acetate enhanced peak (I_p) 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 enhancesI_P via a sequential activation of the focal adhesion kinase cell adhesion kinase β/proline-rich tyrosine kinase 2 (CAKβ/Pyk2) and the nonreceptor tyrosine kinase Src (Huang et al., 1999; Lu et al., 1999). Here, we report that the PKC-induced depression of I_ss is unrelated to the PKC/CAKβ/Src-signaling pathway but depends on the concentration of extracellular Ca²⁺. Intracellular applications of CaM reducedI_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 ofI_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.