Expression of glycine-activated diheteromeric NR1/NR3 receptors in human embryonic kidney 293 cells Is NR1 splice variant-dependent

Abstract

In oocytes, glycine activates receptors formed by diheteromeric combinations of N-methyl-d-aspartate (NMDA) NR1 and NR3 subunits. In contrast, functional receptors in mammalian cells require the simultaneous expression of NR1 and both NR3A and NR3B subunits. In vivo, NR3A and NR3B subunits show differential expression patterns and thus may not naturally form triheteromeric receptors. In this study, we examined whether NR1 splice variants play a role in allowing assembly of functional diheteromeric receptors in mammalian cells. Little current was found in human embryonic kidney 293 cells coexpressing either NR3A or NR3B and the NR1-1a splice variant. However, robust glycine-activated currents were generated in cells transfected with NR3(A or B) and either NR1-2a, NR1-3a, or NR1-4a, and current density was correlated with NR1 C-terminal length. Truncation of the NR1-1a C terminus modestly enhanced NR1-1a/NR3A currents, whereas only small increases were observed with mutations of C-terminal residues that control trafficking or phosphorylation. In contrast, large currents were observed when an extracellular phenylalanine in NR1-1a that influences glycine access was mutated to alanine. A separate mutation in NR1-1a that disrupts glycine binding did not generate responses in NR1-1a/NR3A receptors alone, but it produced a greater than 30-fold potentiation of currents during coapplication of glycine and the glycine antagonist 7-chlorokynurenic acid. Finally, transfection of cells with the NR1-4a subunit along with NR2 and NR3 subunits resulted in the expression of both NR1/NR3 receptors and conventional NMDA receptor currents. These results indicate a prominent role for NR1 splice variants in the functional expression of NR1/NR3 receptors in mammalian cells.

Fig. 1.

Glycine activation of currents from HEK293 cells transfected with NR3A and NR1 splice variants. A, diagram of NR1 splice variants showing alternatively spliced cassettes in the N-terminal domain (N1 cassette, ) and C-terminal domain (C1 cassette, ▥; C2 cassette, ▤; and C2′ cassette, ▨). Gray bars represent transmembrane domains. B, representative traces from NR1-1a/NR3A, NR1-2a/NR3A, NR1-3a/NR3A, and NR1-4a/NR3A recombinant receptors during exposure to 100 μM glycine. The horizontal bar above the traces indicates a 6-s agonist application. Scale bar, 5 s. C, current density relationship for glycine activation of NR1/NR3 receptors containing NR1-1a, NR1-2a, NR1-3a, and NR1-4a splice variants. NR1-1a/NR3A relationship is shown as a dash line for emphasis. The data represent peak current measurements normalized to whole-cell capacitance and expressed as mean ± S.E.M. (n = 9–14 cells per receptor combination). D, concentration-response relationship for glycine activation of NR1/NR3A receptors containing NR1-1a, NR1-2a, NR1-3a, and NR1-4a splice variants. The NR1-1a/NR3A relationship is shown as a dotted line for clarity. Data represent the peak current values expressed as a percentage (mean ± S.E.M.) of that obtained at the maximal glycine concentration (500 μM) tested (n = 10–15 determinations per glycine concentration).

Fig. 2.

Glycine activation of current expression from HEK293 cells transfected with NR3B and NR1 splice variants. A, representative currents from NR1-1a/NR3B, NR1-2a/NR3B, NR1-3a/NR3B, and NR1-4a/NR3B recombinant receptors during exposure (horizontal bar above traces) to indicated micromolar concentration of glycine. Scale bar, 5 s. B, effects of splice variants on NR1/NR3B receptor current density at 10 μM glycine. Data represent the mean ± S.E.M. of 10 μM glycine steady-state current normalized to whole-cell capacitance (n = 3–11 cells). C, normalized glycine concentration-response relationship for NR1-1a/NR3B, NR1-2a/NR3B, NR1-3a/NR3B, and NR1-4a/NR3B recombinant receptors. Data represent the mean ± S.E.M. of steady-state current normalized to whole-cell capacitance and expressed as the ratio to current at 10 μM glycine (I/I₁₀) (n = 6–11 cells per receptor combination). D, current traces from HEK293 cells transfected with NR1-1b/NR3A, NR1-4b/NR3A, NR1-1b/NR3B, and NR1-4b/NR3B. Currents were activated with 100 μM glycine for 6 s as indicated by bars above traces.

Fig. 3.

Effect of NR1 C-terminal domain on NR1/NR3A current expression. A, representative currents from HEK293 cells expressing NR1-4a/NR3A and mutant NR1-1a/NR3A receptors containing a stop codon at residue 863 of the NR1 subunits. Traces from NR1-1a/NR3A receptors are omitted because glycine application does not result in currents from these receptors. Horizontal bars above traces indicate a 6-s application of 100 μM glycine. B, concentration-response relationship of wild-type NR1-1a/NR3A, mutant NR1-1a 863/NR3A, and NR1-4a/NR3A receptors. The data represent peak current measurements normalized to whole-cell capacitance and expressed as mean ± S.E.M. (n = 6–10 cells per receptor combination). C, traces from NR1-4a/NR3A and mutant NR1-1a RRR/NR3A receptors. D, summary graph of concentration-response relationship between NR1-4a/NR3A and mutant NR1-1a RRR/NR3A receptors. Data represent peak current measurements normalized to whole-cell capacitance and expressed as mean ± S.E.M. (n = 5–7 cells per receptor combination). E, traces from NR1-4a/NR3A and mutant NR1-1a S897A-, NR1-1a S897D-, and NR1-1a SS896DD-containing NR1/NR3A receptors. F, summary graph of concentration-response relationship between NR1-4a/NR3A and mutant NR1-1a S897A, NR1-1a S897D and NR1-1a SS896DD receptors. Data represent peak current measurements normalized to whole-cell capacitance and expressed as mean ± S.E.M. (n = 4–5 cells per receptor combination).

Fig. 4.

Effects of NR1 splice variants on membrane surface expression. HEK293 cells were sham transfected (con) or transfected with NR1-1a/NR3 or NR1-4a/NR3A and processed as described in Materials and Methods. The cross-linker (BS³) was used to label the recombinant receptor pool at the surface, and samples were analyzed by Western blot with anti-NR1. A, representative immunoblots of sham-transfected (con) and NR1-1a/NR3A- and NR1-4a/NR3A-transfected HEK293 cells along with corresponding BS³-treated samples. B, densitometry analysis of band intensity from NR1-1a/NR3A- and NR1-4a/NR3A-transfected tissue. Data represent the mean ± S.E.M. of three separate experiments. C, band intensity expressed as the ratio of BS³-treated to corresponding non-BS³-treated sample. Data represent the mean ± S.E.M. of three separate experiments.

Fig. 5.

Influence of glycine sensitivity on NR1/NR3 receptor expression. A, representative traces from NR1-1a F484A/NR3A and NR1-4a F484A/NR3A receptors. B, summary graph of glycine activation of NR1-1a F484A/NR3A and NR1-4a F484A/NR3A receptors. The data represent steady-state current measurements normalized to whole-cell capacitance and expressed as mean ± S.E.M. (n = 3–9 determinations per glycine concentration).

Fig. 6.

Effects of NMDA receptor glycine-site antagonists. A, representative traces of glycine activation of NR1-1a/NR3A and NR1-4a/NR3A receptors in the absence and presence of 100 μM 7-CK. Scale bars, 5 s. B, summary graph of 7-CK potentiation of NR1-1a- and NR1-4a-containing receptors. Data represent the mean ± S.E.M. of the steady-state current from NR1-1a (n = 3 cells) and NR1-4a (n = 7 cells) containing NR3 receptors. C, traces of glycine activation of mutant NR1-1a D732N- and NR1-4a D732N-containing NR3A receptors absence and presence of 100 μM 7-CK. Inset shows glycine activation of NR1-1a D732N/NR3A current. D, summary graph of 7-CK potentiation of NR1-1a D732N- and NR1-4a D732N-containing receptors. Data represent the mean ± S.E.M. of the steady-state current from NR1-1a D732N (n = 7 cells) and NR1-4a D732N (n = 10 cells) containing NR3 receptors.

Fig. 7.

Activation of NR1/NR3A receptors in the presence of NR2A. A, representative traces from HEK293 cells transfected with NR1-4a/NR2A/NR3A or B, NR1-4a/NR2A/NR3B and activated with 10 μM glutamate (Glu) + 100 μM glycine (Gly), Glu alone, Gly alone, Glu + Gly + 100 μM APV, Gly + 100 μM 7-CK, and Gly + 10 μM 5,7-DCK. Traces showing the effect of 7-CK and 5,7-DCK from each respective subunit combination are increased in scale to better show drug action. C, summary of change in steady-state current by 7-CK and 5,7-DCK on NR1–4a/NR2A/NR3A and NR1–4a/NR2A/NR3B recombinant receptors. Data represent the mean ± S.E.M. of the steady-state current measurement from NR1-s4a/NR2A/NR3A (n = 4 cells 7-CK; 4 cells 5,7-DCK) and NR1–4a/NR2A/NR3B (n = 6 cells 7-CK; 3 cells 5,7-DCK) receptors.