ACCELERATED COMMUNICATION Evidence for Direct Protein Kinase-C Mediated Modulation of N-Methyl-D-aspartate Receptor Current

Protein kinase-C (PKC) activation differentially affects currents from N-methyl-D-aspartate (NMDA) type glutamate receptors depending upon their subunit composition. Experiments using chimeras initially indicated that the cytoplasmic C-terminal tails of NR2B (responsive to PKC) and NR2C (unresponsive to PKC) subunits contain the amino acid residues responsible for the observed disparity of PKC effects. However, truncation and point mutation experiments have suggested that PKC action on NMDA receptors may be entirely indirect, working via the phosphorylation of associated proteins. Here we suggest that PKC does, in fact, affect NR2B/NR1–011 NMDA currents by direct phosphorylation of the NR2B tail at residues S1303 and S1323. Replacement of either of these residues with Ala severely reduces PKC potentiation. To verify that S1303 and S1323 are sites of direct phosphorylation by PKC, synthetic peptides from the regions surrounding these sites were used as substrates for in vitro assays with purified rat brain PKC. These results indicate that PKC can directly phosphorylate S1303 and S1323 in the NR2B C terminus, leading to enhanced currents through NMDA receptor channels. The direct action of PKC on certain NMDA receptor subtypes may be important in any physiological or pathological process where PKC and NR2B/NR1 receptors interact. N-Methyl-D-aspartate (NMDA) receptors are a calciumpermeant, depolarization-dependent subtype of ionotropic glutamate receptors that mediates a wide variety of physiological and pathological processes in the central nervous system, including development of proper neuronal circuits (Constantine-Paton et al., 1990), certain learning tasks (Morris et al., 1986; Sakimura et al., 1995), and forms of synaptic modification (Collingridge and Bliss, 1995; Tsien et al., 1996; Ito et al., 1997) and excitotoxicity (Choi, 1988). NMDA receptors are oligomeric complexes generally composed of two types of subunit, the coagonist glycine binding subunit NR1 and the glutamate-binding subunits NR2A–D (Hollmann and Heinemann, 1994). The four NR2 subunits have large cytoplasmic carboxyl termini and are distinct in their functional properties, regulation, and temporal and spatial expression (Hollmann and Heinemann, 1994; Monyer et al., 1994). Protein phosphorylation regulates multiple properties of ligand-gated ion channels, including clustering, desensitization, and peak currents (Swope et al., 1999). Within the NMDA subfamily of glutamate receptors, modulation of peak currents by PKC depends primarily on the NR2 subunits expressed (Kutsuwada et al., 1992). Enhancement by PKC is pronounced for receptors containing the NR2A or NR2B subunits, but absent for receptors containing the NR2C or NR2D subunits (Mori et al., 1993; Wagner and Leonard, 1996). Alternatively spliced variants of the NR1 subunit have more subtle effects on the level of PKC potentiation of currents (Logan et al., 1999). Experiments using chimeras between the NR2B (responsive to PKC) and NR2C (unresponsive to PKC) subunits indicates that the C-terminal region of the NR2B subunit is the critical structural domain for PKCmediated current potentiation (Mori et al., 1993). However, whether the up-regulation of current response is mediated by This work was supported by Grant R01-NS31962–02 from the National Institutes of Health (J.P.L.). 1 Current Address: Department of Physiology, University of Wisconsin, Madison, WI 53706. ABBREVIATIONS: NMDA, N-methyl-D-aspartate; PKC, protein kinase C; kb, kilobase pairs; MOPS, (3-[N-morpholino]propanesulfonic acid); BOS, barium oocyte solution; PDBu, phorbol ester dibutyrate; aa, amino acid(s); PSD-95; postsynaptic density protein 95; CaMK, calmodulin-dependent kinase. 0026-895X/01/5905-960–964$3.00 MOLECULAR PHARMACOLOGY Vol. 59, No. 5 Copyright © 2001 The American Society for Pharmacology and Experimental Therapeutics 632/900017 Mol Pharmacol 59:960–964, 2001 Printed in U.S.A. 960 at A PE T Jornals on A ril 9, 2017 m oharm .aspeurnals.org D ow nladed from PKC phosphorylation of the NR2B C terminus has remained unclear. Although biochemical study has shown that NR2A and NR2B are phosphorylated by PKC (Leonard and Hell, 1997), no specific sites have been defined. Here we report the identification of two important serine residues, S1303 and S1323, in the NR2B C-terminal region that are directly phosphorylated by PKC and control the PKC-mediated enhancement of NR2B/NR1 receptor currents. Materials and Methods Plasmid Construction. Chimeras 1 and 2 were C-terminal chimeras constructed by exchanging the ClaI–NcoI fragment of pBKSAe2 and pBKSAe3, which encodes the mouse NR2B and NR2C subunits, respectively, as described in Mori et al. (1993). Chimeras 3 to 5 were three mutant NR2B subunits containing different regions of the NR2B C terminus and part of the NR2C C terminus (Fig. 1). The fragments used to construct these chimeras were, for chimera 3, the 3.8-kb ClaI-Eco47III fragment of NR2B and the 4.0-kb Eco47IIIClaI fragment of NR2C; for chimera 4, the 4.1-kb ClaI-StuI fragment of NR2B and the 4.0-kb EheI-ClaI fragment of NR2C; and, for chimera 5, the 6.4-kb SpeI-EheI fragment of chimera 1 and the 1.3-kb StuI-SpeI fragment of NR2B. Deletions 1 and 2 were in-frame, Cterminal deletion mutants. Deletion 1 was constructed by deleting a 1008-base-pair Bpu1102 I fragment. Deletion 2 was made by deleting a 214-base-pair EcoNI fragment followed by a fill-in. Site-specific mutants S1303A, S1323A, S1354A, the double and triple mutants, were constructed by polymerase chain reaction mutagenesis using Pfu polymerase and confirmed by dideoxynucleotide sequencing. RNA Preparation and Injection. Plasmids pBKSAz1, -e2, and -e3 containing cDNAs encoding mouse NMDA receptor subunits NR1011, NR2B, and NR2C, respectively were kindly provided by Dr. Masayoshi Mishina (University of Tokyo, Japan). Complementary RNAs were transcribed in vitro using T3 RNA polymerase with SpeI linearized templates. Xenopus laevis oocytes were prepared as described previously (Liao and Leonard, 1999) and injected with 10 ng of RNAs in a 1:1 M ratio of NR1 to NR2 wild-type or mutant