Pharmacological characterization of recombinant NR1/NR2A NMDA receptors with truncated and deleted carboxy termini expressed in Xenopus laevis oocytes

Abstract

Background and purpose: The carboxy terminal domain (CTD) of NR2 N-methyl-d-aspartate receptor (NMDAR) subunits interacts with numerous scaffolding and signal transduction proteins. Mutations of this region affect trafficking and downstream signalling of NMDARs. This study determines to what extent characteristic pharmacological properties of NR2A-containing NMDARs are influenced by this key functional domain.

Experimental approach: Using recombinant receptor expression in Xenopus laevis oocytes and two electrode voltage clamp recordings we characterized pharmacological properties of rat NR1/NR2A NMDARs with altered CTDs. We assessed the effects of truncating [at residue Iso1098; NR2A(trunC)] and deleting [from residue Phe822; NR2A(delC)] the CTD of NR2A NMDAR subunits on agonist potencies, channel block by Mg(2+) and memantine and potentiation of NMDAR-mediated responses by chelating contaminating divalent cations.

Key results: Truncation or deletion of the CTD of NR2A NMDAR subunits did not affect glutamate potency [EC(50) = 2.2 micromol.L(-1), NR2A(trunC); 2.7 micromol.L(-1), NR2A(delC) compared with 3.3 micromol.L(-1), NR2A(WT)] but did significantly increase glycine potency [EC(50) = 500 nmol.L(-1), NR2A(trunC); 900 nmol.L(-1), NR2A(delC) compared with 1.3 micromol.L(-1), NR2A(WT)]. Voltage-dependent Mg(2+) block of NR2A(WT)- and NR2A(trunC)-containing NMDARs was similar but low concentrations of Mg(2+) (1 micromol.L(-1)) potentiated NR1/NR2A(delC) NMDARs. Memantine block was not affected by changes to the structure of the NR2A CTD. EDTA-induced potentiation was similar at each of the three NMDAR constructs.

Conclusions and implications: Of the parameters studied only minor influences of the CTD were observed; these are unlikely to compromise interpretation of studies that make use of CTD-mutated recombinant receptors or transgenic mice in investigations of the role of the CTD in NMDAR signalling.

Figure 1

Representation of the NR2A NMDAR subunits used in this study. (A) Cartoon representation of an NMDAR subunit showing the proposed membrane topology of three membrane spanning domains (M1, M3 and M4) and a re-entrant loop (M2) and the location of the amino terminal domain (ATD) and carboxy terminal domain (CTD). The ligand binding domains (denoted D1 and D2) are formed by the S1 and S2 regions of the protein which come together to form a hinged clamshell-like structure. (B) Amino acid sequence of part of the NR2A NMDAR subunit. The letters shown in bold indicate the M4 region while each of the residues labelled as i, ii and iii indicate the last amino acid contained within each of the subunits studied. The full-length NR2A(WT) subunit contains 1145 residues and ends at Val1445 (i); the NR2A(trunC) NMDAR subunit ends at Iso1098 (ii) while the NR2A(delC) NMDAR subunit contains only four residues of the carboxy terminal and ends at Phe822 (iii). (C) Cartoon depiction and linear representation of the structure of each of the three NR2A NMDAR subunits investigated in this study.

Figure 2

Concentration-response curves for NR1/NR2A(trunC) and NR1/NR2A(delC) NMDARs. (A) Example of a TEVC current recording obtained from an oocyte expressing NR1/NR2A(delC) NMDARs. In the presence of glycine (50 µmol·L⁻¹), increasing concentrations of glutamate (100 nmol·L⁻¹–300 µmol·L⁻¹) were applied cumulatively. (B) Mean concentration-response curve for glutamate-evoked currents recorded from oocytes expressing NR1/NR2A(trunC) NMDARs. The data points are fitted with the Hill equation, which gives an EC₅₀ value of 2.5 µmol·L⁻¹ (n = 8). (C) Mean concentration-response curve for glutamate-evoked currents recorded from oocytes expressing NR1/NR2A(delC) NMDARs, the EC₅₀ is 3.2 µmol·L⁻¹ (n = 12). (D) As in (A) but showing a TEVC current trace obtained in the presence of glutamate (100 µmol·L⁻¹) and the cumulative addition of increasing concentrations of glycine (100 nmol·L⁻¹–300 µmol·L⁻¹). (E) Mean concentration-response curve for glycine acting at NR1/NR2A(trunC) NMDARs, the estimated EC₅₀ value is 0.46 µmol·L⁻¹ (n = 6). (F) Mean concentration-response curve for glycine-evoked currents recorded from oocytes expressing NR1/NR2(delC) NMDARs, the EC₅₀ is 1.4 µmol·L⁻¹ (n = 6). The mean maximal currents recorded for glutamate concentration-response curves were 3.1 ± 1.0 µA and 7.9 ± 0.7 µA and for glycine concentration-response curves were 5.0 ± 0.7 µA and 5.1 ± 0.7 µA for oocytes expressing NR1/NR2A(trunC) and NR1/NR2A(delC) NMDARs respectively. The dashed lines in each of the panels indicate the mean concentration-response curves for glutamate or glycine acting at NR1/NR2A(WT) NMDARs. Data originally presented in Erreger et al. (2007) for (B) and (C) and in Chen et al. (2008) for (E) and (F).

Figure 3

Mg²⁺ inhibition of NR1/NR2A(WT), NR1/NR2A(trunC) and NR1/NR2A(delC) NMDARs. (A) TEVC current trace, at −80 mV, of glutamate-evoked current recorded from an oocyte expressing NR1/NR2A(WT) NMDARs. NMDAR-mediated currents are shown in the absence (control) and presence of increasing concentrations of Mg²⁺ (1 µmol·L⁻¹–1 mmol·L⁻¹). (B) As (A) but obtained from an oocyte expressing NR1/NR2A(trunC) NMDARs. (C) TEVC trace, recorded at −40 mV, illustrating the novel finding of Mg²⁺-induced potentiation, at low Mg²⁺ concentrations, of NR1/NR2A(delC) NMDAR-mediated currents. (D–F) Histograms showing the mean percentage inhibition of glutamate-evoked currents by Mg²⁺ for oocytes voltage-clamped at −80, −60 and −40 mV.

Figure 4

Memantine inhibition of NR1/NR2A(WT), NR1/NR2A(trunC) and NR1/NR2A(delC) NMDARs. (A) TEVC current trace, at −80 mV, of glutamate-evoked current recorded from an oocyte expressing NR1/NR2A(WT) NMDARs. NMDAR-mediated currents are shown in the absence (control) and presence of increasing concentrations of memantine (0.1–30 µmol·L⁻¹). (B and C) As in (A) but obtained from oocytes expressing NR1/NR2A(trunC) NMDARs (B) or NR1/NR2A(delC) NMDARs (C). (D–F) Histograms showing the mean percentage inhibition of glutamate-evoked currents by memantine for oocytes voltage-clamped at −80, −60 and −40 mV.

Figure 5

EDTA-induced potentiation of NR1/NR2A(WT), NR1/NR2A(trunC) and NR1/NR2A(delC) NMDARs. (A) TEVC current trace, at −40 mV, of glutamate-evoked current recorded in a low BaCl₂ (0.18 mmol·L⁻¹) external solution. Once a steady-steady response was obtained, the solution was switched to one containing EDTA (10 µmol·L⁻¹). This resulted in an increase in the level of current recorded due to the chelation of contaminant levels of certain divalent cations (mainly Zn²⁺). (B and C) As (A) but illustrating TEVC traces obtained from oocytes expressing NR1/NR2A(trunC) NMDARs (B) or NR1/NR2A(delC) NMDARs (C). (D) Histogram showing the mean potentiation of glutamate-evoked currents by EDTA. No significant differences in the levels of potentiation were observed between each of the three NR2A constructs.