Mg2+ and memantine block of rat recombinant NMDA receptors containing chimeric NR2A/2D subunits expressed in Xenopus laevis oocytes

Abstract

N-methyl-d-aspartate receptors (NMDARs) display differences in their sensitivity to the channel blockers Mg(2+) and memantine that are dependent on the identity of the NR2 subunit present in the receptor-channel complex. This study used two-electrode voltage-clamp recordings from Xenopus laevis oocytes expressing recombinant NMDARs to investigate the actions of Mg(2+) and memantine at the two NMDARs displaying the largest differences in sensitivity to these blockers, namely NR1/NR2A and NR1/NR2D NMDARs. In addition, NR2A/2D chimeric subunits have been employed to examine the effects of pore-forming elements and ligand-binding domains (LBD) on the potency of the block produced by each of these inhibitors. Our results show that, as previously documented, NR2D-containing NMDARs are less sensitive to voltage-dependent Mg(2+) block than their NR2A-containing counterparts. The reduced sensitivity is determined by the M1M2M3 membrane-associated regions, as replacing these regions in NR2A subunits with those found in NR2D subunits results in a approximately 10-fold reduction in Mg(2+) potency. Intriguingly, replacing the NR2A LBD with that from NR2D subunits results in a approximately 2-fold increase in Mg(2+) potency. Moreover, when responses mediated by NR1/NR2A NMDARs are evoked by the partial agonist homoquinolinate, rather than glutamate, Mg(2+) also displays an increased potency. Memantine block of glutamate-evoked currents is most potent at NR1/NR2D NMDARs, but no differences are observed in its ability to inhibit NR2A-containing or NR2A/2D chimeric NMDARs. We suggest that the potency of block of NMDARs by Mg(2+) is influenced not only by pore-forming regions but also the LBD and the resulting conformational changes that occur following agonist binding.

Figure 1. Cartoon illustration of an NMDAR subunit and the various chimeric constructs examined

A, cartoon sketch of an ionotropic glutamate receptor 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, linear representation of the various NMDAR constructs investigated and the nomenclature used in this study. Regions originating from the NR2A subunit are shown in grey, while those originating from the NR2D subunit are shown in white. C, cartoon representation of these constructs showing how the various functional domains from the NR2D subunit are incorporated into the three chimeric subunits.

Figure 2. TEVC current recordings illustrating voltage and concentration dependence of Mg²⁺ block in wild-type and chimeric NMDARs

A, example TEVC traces recorded from an oocyte expressing NR1/NR2A NMDARs The glutamate-evoked currents recorded at each of the membrane potentials indicated are reversibly inhibited by coapplication of Mg²⁺ (100 μm) at the point indicated by the bar. B, same traces as illustrated in A but with the glutamate-evoked current recorded in the absence of Mg²⁺ at −60 mV and −40 mV scaled to equal the steady-state current recorded at −80 mV. The extent of the inhibition decreases at more depolarized membrane potentials. C, TEVC traces recorded from an oocyte expressing NR1/NR2A NMDARs and voltage-clamped at either −80 mV or −40 mV. The black bar at the top of the trace in this panel and panels D and E below indicates the duration of the bath application of glutamate (3 μm), while the shaded bar in this panel (and those below) indicates the coapplication of Mg²⁺. Increasing concentrations of Mg²⁺ were applied, cumulatively, as indicated by the arrowheads. D, TEVC traces recorded from an oocyte expressing NR1/NR2D NMDARs and voltage-clamped at either −80 mV or −40 mV illustrating the block of the glutamate-evoked current by increasing concentrations of Mg²⁺. Note that NR1/NR2D NMDAR-mediated currents are less sensitive to block by Mg²⁺ compared to responses mediated by NR1/NR2A NMDARs. E, TEVC traces recorded from an oocyte expressing NR1/NR2A(2D-S1S2) NMDARs and voltage-clamped at either −80 mV or −40 mV illustrating the block of the glutamate-evoked current by increasing concentrations of Mg²⁺. Note that NR1/NR2A(2D-S1S2) NMDAR-mediated currents are more sensitive to block by Mg²⁺ compared to responses mediated by NR1/NR2A NMDARs.

Figure 3. Mean inhibition curves for Mg²⁺ block of glutamate-evoked currents mediated by wild-type and chimeric NMDARs

A, mean inhibition curves for Mg²⁺ block of glutamate-evoked NR1/NR2A NMDAR-mediated currents. Inhibition curves were constructed at −80 mV (▪), −60 mV (•) and −40 mV (▴) and fitted with the Hill equation (see Methods). B, mean inhibition curves for Mg²⁺ block of NR1/NR2D NMDAR-mediated currents. C, mean inhibition curves for Mg²⁺ block of NR1/NR2A(2D-S1S2) NMDAR-mediated currents. D, mean inhibition curves for Mg²⁺ block of NR1/NR2A(2D-M1M2M3) NMDAR-mediated currents. E, mean inhibition curves for Mg²⁺ block of NR1/NR2A(2D-S1M1M2M3S2) NMDAR-mediated currents. Mean IC₅₀ values determined at each holding potential for each construct are given in Table 1.

Figure 4. Mean inhibition curves for Mg²⁺ block of homoquinolinate-evoked currents mediated by wild-type and chimeric NMDARs

A, mean inhibition curves for Mg²⁺ block of homoquinolinate-evoked NR1/NR2A NMDAR-mediated currents. Inhibition curves were constructed at −80 mV (▪), −60 mV (•) and −40 mV (▴) and fitted with the Hill equation (see Methods). B, mean inhibition curves for Mg²⁺ block of NR1/NR2D NMDAR-mediated currents. C, mean inhibition curves for Mg²⁺ block of NR1/NR2A(2D-S1S2) NMDAR-mediated currents. D, mean inhibition curves for Mg²⁺ block of NR1/NR2A(2D-M1M2M3) NMDAR-mediated currents. E, mean inhibition curves for Mg²⁺ block of NR1/NR2A(2D-S1M1M2M3S2) NMDAR-mediated currents. Mean IC₅₀ values determined at each holding potential for each construct are given in Table 1.

Figure 5. TEVC current recordings illustrating block by memantine of glutamate-evoked responses mediated by NR2A- and NR2D-containing NMDARs

A, TEVC trace recorded from an oocyte expressing NR1/NR2A NMDARs and voltage-clamped at −80 mV. The black bar at the top of the trace in this panel and panels B and C below indicates the duration of the bath application of glutamate (3 μm). The reversibility of memantine block is illustrated by coapplying with glutamate, memantine at two concentrations (100 nm or 10 μm). Switching the memantine concentration from 10 μm to 100 nm results in a recovery of the glutamate-evoked current to the level seen when memantine was first applied. B, TEVC traces recorded from an oocyte expressing NR1/NR2A NMDARs and voltage-clamped at either −80 mV or −40 mV. The shaded bar in this panel (and in panel C below) indicates the coapplication memantine. Increasing concentrations of memantine were applied, cumulatively, as indicated by the arrowheads. C, TEVC traces recorded from an oocyte expressing NR1/NR2D NMDARs and voltage-clamped at either −80 mV or −40 mV illustrating the block of the glutamate-evoked current by increasing concentrations of memantine. Note that NR1/NR2D NMDAR-mediated currents are more sensitive to block by memantine compared to responses mediated by NR1/NR2A NMDARs.

Figure 6. Mean inhibition curves for memantine block of glutamate-evoked currents mediated by wild-type and chimeric NMDARs

A, mean inhibition curves for memantine block of glutamate-evoked NR1/NR2A NMDAR-mediated currents. Inhibition curves were constructed at −80 mV (▪), −60 mV (•) and −40 mV (▴) and fitted with the Hill equation (see Methods). B, mean inhibition curves for Mg²⁺ block of NR1/NR2D NMDAR-mediated currents. C, mean inhibition curves for Mg²⁺ block of NR1/NR2A(2D-S1S2) NMDAR-mediated currents. D, mean inhibition curves for Mg²⁺ block of NR1/NR2A(2D-M1M2M3) NMDAR-mediated currents. E, mean inhibition curves for Mg²⁺ block of NR1/NR2A(2D-S1M1M2M3S2) NMDAR-mediated currents. Mean IC₅₀ values determined at each holding potential for each construct are given in Table 1.

Figure 7. Voltage dependence of IC₅₀ values for Mg²⁺ and memantine block of wild-type and chimeric NMDAR-mediated responses and comparison of δ values

A–E, plots of the voltage dependence of the mean IC₅₀ value for Mg²⁺ block of glutamate-evoked (□) or homoquinolinate-evoked (○) currents and memantine block of glutamate-evoked currents (▵) in oocytes expressing NR1/NR2A NMDARs (A), NR1/NR2D NMDARs (B), NR1/NR2A(2D-S1S2) NMDARs (C), NR1/NR2A(2D-M1M2M3) NMDARs (D) or NR1/NR2A(2DS1M1M2M3S2) NMDARs (E). Note that for each plot the slope of the line describing memantine block of the currents is shallower than that seen for Mg²⁺ block of the same NMDAR-mediated current, though the potency of memantine is greater than that of Mg²⁺. F–H, bar graphs showing δ values determined from analysis of Mg²⁺ block of glutamate-evoked currents (F), Mg²⁺ block of homoquinolinate-evoked currents (G) and memantine block of glutamate-evoked currents (H). Mean δ values for each of the blockers are indicated.