Pharmacological Characterization of Glycine-Activated Currents in HEK 293 Cells Expressing N-Methyl-D-aspartate NR1 and NR3 Subunits

Abstract

N-Methyl-D-aspartate (NMDA) receptors are important targets for drugs of abuse such as ethanol, toluene, and ketamine. Ligand-gated ion channels assembled from the NR1 and NR3 subunits have functional and pharmacological properties that are distinct from those of conventional NMDA receptors containing NR2 subunits. In the present study we used voltageclamp electrophysiology to characterize excitatory glycine-activated receptors assembled from NR1, NR3A, and NR3B subunits expressed in human embryonic kidney (HEK) 293 cells. These glycine-activated receptors were not stimulated by glutamate or kainic acid and were resistant to magnesium block. A wide variety of NMDA receptor antagonists including D-2-amino-5-phosphonovaleric acid, ifenprodil, memantine, (5R,10S)-(ϩ)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801) or acamprosate did not inhibit glycine-activated NR1/NR3A/NR3B receptors. Likewise, these receptors were not affected by antagonists of inhibitory glycine receptors or glycine transporters. The NMDA receptor glycine site agonist, D-serine, partially activated NR1/ NR3A/NR3B receptors, whereas the antagonist, 5,7-dichlorokynurenic acid, inhibited receptor currents. Conversely, the antagonist, 7-chlorokynurenic acid, and the partial agonist, R-(ϩ)-3-amino-1-hydroxy-2-pyrrolidinone (HA-966), potentiated glycine-stimulated currents of these receptors. NR1/ NR3A/NR3B receptor currents were inhibited by 10 to 21% by ethanol and toluene but were relatively insensitive to ketamine. Ethanol inhibition was enhanced in receptors expressing the NR1(L819A) mutant, whereas those containing NR1(F639A) or NR1(M813A) showed no change relative to the wild-type NR1. The results of this study indicate that coexpression of NR1, NR3A, and NR3B subunits in HEK 293 cells results in glycineactivated receptors with novel functional and pharmacological properties.N-Methyl-D-aspartate (NMDA) receptors are glutamateactivated ion channels that contain NR1 and NR2 subunits. A third class of NMDA receptor subunits has also been reported and consists of the NR3A and NR3B subunits (Ciabarra et al., 1995;Sun et al., 1998). NR3 subunits share structural features with the NR1 subunit, including the S1 and S2 agonist binding domains and some sequence similarity across transmembrane domains (Chatterton et al., 2002). Coexpression of the NR3A or NR3B subunits with NR1 and NR2 in recombinant expression systems decreases NMDA receptor current (Chatterton et al., 2002) and reduces calcium permeability (Matsuda et al., 2002). In NR3A-deficient mice, NMDA receptor currents are significantly enhanced (Das et al., 1998), suggesting that these subunits may be important modulators of NMDA receptor function.As for NR1 subunits, NR3 subunits do not form functional channels when expressed alone. However, coexpression of NR1 and NR3 subunits in oocytes has been reported to produce functional channels that are gated by glycine but not glutamate (Chatterton et al., 2002; but see Smothers and Woodward, 2003). In...

New Insights into the Not-So-New NR3 Subunits of N-Methyl-d-aspartate Receptor: Localization, Structure, and Function

“…NR3A subunit shows absolute selectivity for glycine over glutamate. These biochemical data corroborated with functional electrophysiological observations that showed glycine alone is sufficient to activate heterologous diheteromeric NR1/NR3 and triheteromeric NR1/ NR3A/NR3B receptors (Chatterton et al, 2002;Smothers and Woodward, 2007) (Fig. 4).…”

Rules of engagement for NMDA receptor subunits

“…In contrast, there is evidence that the triheteromeric NR1/NR2/NR3 receptor forms in parietooccipital cortical neurons of P8 mice, based on the observation of single-channel currents with reduced amplitude that were induced by NMDA, were Mg 2ϩ -insensitive, and could be blocked by APV, properties that could not be attributed to either NR1/NR2 or NR1/NR3 receptors (16). In HEK293 cells, upon coexpression of one type of NR3 (NR3A or NR3B) with NR1/NR2, the NMDA-induced Ca 2ϩ permeability of the cells changed and channels with reduced conductance were observed, also supporting the formation of NR1/NR2/NR3 triheteromers because NR1/NR3A or NR1/NR3B receptors do not form in HEK293 cells (12,14,17). It is noteworthy, however, that although coexpression of NR1/ NR2/NR3 appears to consistently result in reduced whole-cell current when compared with expression of NR1/NR2 in both oocytes and HEK 293 cells, Sucher et al (9) failed to detect a change in the Mg 2ϩ sensitivity of NMDA-or glutamate-induced currents in oocytes and Nishi et al (11) failed to do so in HEK 293 cells.…”

Glutamate Receptor Ion Channels: Structure, Regulation, and Function

“…Occupancy of the GluN1 subunit by ligand within GluN1/GluN3 receptors seems to facilitate desensitization. In GluN1/GluN3 receptors, GluN3 is activated fully by glycine and partially by D-serine and 1-aminocyclopropane-1-carboxylic acid (Chatterton et al, 2002;Smothers and Woodward, 2007), which may account for observations that D-serine antagonized GluN1/GluN3 receptors (Awobuluyi et al, 2007).…”

“…When coexpressed with GluN1 in X. laevis oocytes, GluN1/GluN3 receptors can form receptors that are activated by glycine alone (Chatterton et al, 2002), but these excitatory glycine receptors have not yet been observed in GluN3-expressing neurons (Matsuda et al, 2003). At present, surface expression of glycine-activated GluN1/GluN3A or GluN1/GluN3B receptors in HEK293 cells is unresolved, but GluN1/ GluN3A/GluN3B shows some functional expression (Smothers and Woodward, 2007). When GluN3 is coexpressed with GluN1 and GluN2 in X. laevis oocytes, NMDA-and glutamate-activated current amplitudes are reduced compared with current from GluN1/GluN2, suggesting that either triheteromeric GluN1/GluN2/ GluN3 receptors form that have a lower conductance, or GluN3 expression reduces trafficking or assembly of GluN1/GluN2 (Das et al, 1998;Perez-Otano et al, 2001; Isacoff, 2007, 2008).…”