Opposing Contributions of NR1 and NR2 to Protein Kinase C Modulation of NMDA Receptors

Abstract

N‐Methyl‐d‐aspartate (NMDA) receptors mediate increases in intracellular calcium that can be modulated by protein kinase C (PKC). As PKC modulation of NMDA receptors in neurons is complex, we studied the effects of PKC activation on recombinant NMDA receptor‐mediated calcium rises in a nonneuronal mammalian cell line, human embryonic kidney 293 (HEK‐293). Phorbol 12‐myristate 13‐acetate (PMA) pretreatment of HEK‐293 cells enhanced or suppressed NMDA receptor‐mediated calcium rises based on the NMDA receptor subunit composition. NR2A or NR2B, in combination with NR1011, conveyed enhancement whereas NR2C and NR2D conveyed suppression. The PKC inhibitor bisindolylmaleimide blocked each of these effects. The region on NR2A that conveyed enhancement localized to a discrete segment of the C terminus distal to the portion of NR2C that is homologous to NR2A. Calcium‐45 accumulation, but not intracellular calcium store depletion, matched PMA effects on NMDA receptor‐mediated calcium changes, suggesting that these effects were not due to effects on intracellular calcium stores. The suppression of intracellular calcium transients seen with NR2C was eliminated when combined with NR1 splice variants lacking C‐terminal cassette 1. Thus, the intracellular calcium effects of PMA were distinguishable based on both the NR1 splice variant and the NR2 subunit type that were expressed. Such differential effects resemble the diversity of PKC effects on NMDA receptors in neurons.

Proteolysis of theN-Methyl-d-Aspartate Receptor by Calpain in Situ

“…Using HEK293t cells, the transfection efficiency is ϳ70%. As previously shown, individual HEK293t cells express all of the proteins that the cDNAs encode regardless of the number of different cDNA plasmids that are transfected (Grant et al, 1998).…”

“…HEK293t cells were grown on tissue culture-grade dishes (Corning brand; Corning Glassworks, Corning, NY) in RPMI media containing 5% horse serum and 5% fetal bovine serum supplemented with 2 mM glutamine and 100 units/ml penicillin/streptomycin and placed in a 5% CO 2 incubator at 37°C. Transfection of HEK293t cells with cDNA was accomplished by calcium phosphate precipitation as previously described (Grant et al, 1998). Twenty-four hours after transfection, the medium was changed and treatments were added.…”

“…The shortened NR2A subunit NR2A1051 was constructed as previously described (Grant et al, 1998). This construct does maintain the epitope tag required for immunoreactivity to antibody AB1548, consisting of the last six amino acids of the NR2A subunit, and has normal electrophysiological and ligand-binding properties (Grant et al, 1998;Guttmann et al, 2001).…”

“…These interactions can lead to subtype-specific modulation of the receptor. For example, yotiao and protein kinase C modulate NMDA receptors in subunit-specific manners based on the properties of the C-terminal region (Grant et al, 1998;Lin et al, 1998). The importance of the C-terminal region in proper NMDA receptor function is further exemplified by the findings of Sprengel et al (1998), which demonstrated that gene-targeted mice lacking the C-terminal tail of the NR2 subunit exhibited properties similar to mice with a complete absence of an NR2 subunit.…”

Protein kinases and light: unlikely partners in a receptor localization puzzle

“…We then evaluated whether recombinant NMDA receptors assembled by an NR2A subunit truncated at its c-terminus, previously shown to be insensitive to phosphorylation [11], would be less sensitive to light. Unexpectedly, receptors assembled by NR1a/NR2AD 880 were equally sensitive to light modulation, when compared to wild-type receptors (1.7 ± 0.2-fold potentiation, n = 5).…”