A subunit-selective potentiator of NR2C- and NR2D-containing NMDA receptors

Abstract

NMDA receptors are tetrameric complexes of NR1 and NR2A-D subunits that mediate excitatory synaptic transmission and have a role in neurological disorders. In this article, we identify a novel subunit-selective potentiator of NMDA receptors containing the NR2C or NR2D subunit, which could allow selective modification of circuit function in regions expressing NR2C/D subunits. The substituted tetrahydroisoquinoline CIQ (3-chlorophenyl)(6,7-dimethoxy-1-((4-methoxyphenoxy)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)methanone) enhances receptor responses two-fold with an EC(50) of 3 μM by increasing channel opening frequency without altering mean open time or EC(50) values for glutamate or glycine. The actions of CIQ depend on a single residue in the M1 region (NR2D Thr592) and on the linker between the N-terminal domain and agonist binding domain. CIQ potentiates native NR2D-containing NMDA receptor currents from subthalamic neurons. Our identification of a subunit-selective NMDA receptor modulator reveals a new class of pharmacological tools with which to probe the role of NR2C- and NR2D-containing NMDA receptors in brain function and disease.

Figure 1

CIQ selectively potentiates NR2C and NR2D subunits. a. Two-electrode voltage-clamp recordings of recombinant NMDA receptors expressed in Xenopus oocytes and activated by 100 μM glutamate plus 30 μM glycine in the absence and presence of increasing concentrations of CIQ (1 – 100 μM). b. Concentration-response curves show the subunit-selectivity of CIQ (n = 18-21 oocytes per receptor). The response to 100 μM glutamate and 30 μM glycine in the absence of CIQ is normalized to 100%. The structure of CIQ is shown at the right. The dashed box highlights the concentration (10 μM) of CIQ that produces potentiation of responses from NR1/NR2C (●) and NR1/NR2D (■) without affecting responses from NR1/NR2A (□), NR1/NR2B (○), AMPA (GluR1, △), and kainate (GluR6, ▽) receptors. c. The responses of wild type diheteromeric NMDA receptors to 10 μM CIQ plus 100 μM glutamate and 30 μM glycine are shown as a percentage of the response in the absence of CIQ (100%). The responses of NR1/NR2C and NR1/NR2D in the presence of CIQ were significantly different than in control (* p<0.05; paired t-test; n = 6 - 14). The responses of triheteromeric NMDA receptors containing NR2A(N614K,T690I) (hereafter NR2A*) to 10 μM CIQ in the presence of 10 mM glutamate, 100 μM glycine, and 1 mM Mg²⁺ are shown as a percentage of the response in the absence of CIQ (100%). While triheteromeric receptors composed of NR1/NR2A*/NR2B were not potentiated by CIQ, NR1/NR2A*/NR2C, and NR1/NR2A*/NR2D triheteromeric receptors were significantly potentiated by CIQ (* p<0.05; paired t-test; n = 8 - 18). d. The responses of GluR1, GluR2, GluR3, GluR4, GluR5, and GluR6 receptors to 100 μM glutamate in the presence of 10 μM CIQ are not significantly different than control (n = 4 – 10); oocytes expressing GluR5 and GluR6 were exposed to 10 μM concanavalin-A for 10 minutes prior to recording. Responses of GluR6/KA2 receptors were evoked by 100 μM AMPA. For all panels, values are mean ± s.e.m.

Figure 2

CIQ selectively potentiates NR1/NR2C and NR1/NR2D receptors in HEK 293 cells. a. Representative whole-cell voltage-clamp recordings of NR1/NR2C and NR1/NR2D at 23°C. CIQ (10 μM in 100 μM glutamate and 30 μM glycine) evokes 180% responses from both NR1/NR2C and NR1/NR2D compared to control (100 μM glutamate and 30 μM glycine). b. The concentration-effect relationships demonstrate CIQ selectively potentiates NR1/NR2C (●, EC₅₀ = 1.7 μM; n = 5-11) and NR1/NR2D (■, EC₅₀ = 4.1 μM; n = 4-17) to 180% in mammalian cells, but has no effect on NR1/NR2A (□, n = 4-15). CIQ causes a modest (25%) reduction in the NR1/NR2B response at 20 μM (○, n = 5). CIQ (10 μM) potentiated the response of NR1/NR2D to NMDA at 33°C by 200±20% (n = 5, × on plot). Values are mean ± s.e.m. c. Current responses of NR1/NR2C (○) and NR1/NR2D (●) receptors to 100 μM glutamate and 30 μM glycine (white bar) and concurrent application of 10 μM CIQ (gray bar) show that the de-potentiation time course is described by a single exponential function (white line; ). d. The de-potentiation time course is independent of CIQ concentration for NR1/NR2C (n = 3) and NR1/NR2D receptors (n = 6). Values are mean ± s.e.m. e. Unitary currents from a representative outside-out patch containing NR1/NR2D receptors activated by 1 mM glutamate and 50 μM glycine in the absence or presence of 10 μM CIQ. The boxed region is expanded below; c denotes the closed and o the open level. f. Composite distributions of the contiguous open period durations from 6 patches can be fitted by two exponential components with time constants of 0.044 ms and 0.57 ms for control and 0.036 ms and 0.63 ms for CIQ (see also Table 1). The composite closed duration histogram constructed from recordings in the same 6 patches can be fitted by the sum of 6 exponential components, with time constants (area in parentheses) of 0.022 (29), 0.19 (14), 1.7 (12), 5.5 (27), 19 (17) and 94 (0.4) ms in the absence and 0.026 (37), 0.23 (18), 0.73 (12), 3.4 (18), 12 (15) and 39 (0.75) ms in the presence of 10 μM CIQ.

Figure 3

Structural determinants for transferring CIQ sensitivity to NR2A a. Schematic representation of the NR2 subunit polypeptide illustrates regions comprising the amino-terminal domain (ATD), the agonist binding domain (two segments of the polypeptide chain called S1 and S2), a membrane-associated domain that forms the ion channel pore and contains three membrane-spanning helices (M1, M3, M4) with a re-entrant loop (M2), and an intracellular C-terminal domain. Schematic representations of chimeras between the NR2D ATD-M1 region and the corresponding region in NR2A are shown (see Supplementary Table S4 for chimeric junctions). b. The response to 10 μM CIQ plus 100 μM glutamate and 30 μM glycine is shown as a percentage of the response in the absence of CIQ. CIQ sensitivity is observed (p<0.05 compared to wild type NR2A; one-way ANOVA with a Dunnett’s post test) for chimeras 2A-(2D L+S1M1M2M3S2), 2A-(2D L+S1M1), 2A-(2D L+S1M1b), 2A-(2D L+S1M1c), and 2A-(2D L+M1e), suggesting transfer of CIQ sensitivity to NR2A requires residues 590-594 from NR2D and the NR2D linker region (L) between the ATD and the S1 region. Results are from 4-23 oocytes per receptor tested. Values are mean ± s.e.m. c. Model of an NR1/NR2 heterodimer of the tetrameric NMDA receptor based on the GluR2 structure is shown. M4 and the C-terminal domain are omitted for clarity. The NR1 and NR2 subunits are shown in yellow and brown, respectively. The regions that together can transfer CIQ sensitivity from NR2D to NR2A (T592 and the ATD-S1 linker) are shown as grey spheres and highlighted in black circles.

Figure 4

Structural determinants of CIQ action in NR2D a. Alignment of amino acid sequences for the ATD-S1 linker and M1 regions of NR2A-D. Fully conserved residues are shown in yellow, and residues conserved only in NR2C and NR2D are shown in green. Arrows mark residues that were converted in NR2D from the NR2C/D residue to the NR2A/B residue and vice versa by site-directed mutagenesis (Supplementary Table S4). b. Four M1 residues in NR2D that are conserved between NR2C/D but not NR2A/B were mutated to the corresponding residue in NR2A/B. Responses of the mutant receptor NR1/NR2D(T592I) were no longer potentiated by CIQ. Chimeric NR2D receptors with the ATD+L showed a reduced level of potentiation. Results are from 6-10 oocytes per receptor tested. *, p<0.05 compared to NR1/NR2D, one-way ANOVA with a Tukey’s post hoc test). Values are mean ± s.e.m.

Figure 5

CIQ potentiates native NR2D-containing NMDA receptors in subthalamic neurons a. Photomicrograph of the experimental setup for whole cell voltage-clamp recording from subthalamic (STN) neurons. A pressurized (4-12 psi; 3-100 ms application) micropipette (3.5 MOhm) was used to apply glycine (0.5-1 mM) and NMDA (1-2 mM) to the subthalamic neuron, which was held at −60 mV. Lower right, neurons demonstrated characteristic I_h current, evoked by injecting 0.1 nA hyperpolarizing current into the cell. b. CIQ (20 μM) potentiated STN neurons to 230±29% (n = 6; white bars) compared to the control response. Potentiation by CIQ was reversible, with NMDA-activated responses after recovery being 102±11% of control amplitude. NMDA-activated responses could be inhibited by the NMDA receptor competitive antagonist D,L-APV (200-400 μM; 27±7% residual current compared to control). CIQ did not potentiate the current response to pressure-applied NMDA in hippocampal CA1 pyramidal neurons (n = 5; black bars). Data are mean ± s.e.m. * p<0.01; one-way ANOVA with repeated measures and Tukey’s post hoc test. c. Representative whole cell-voltage clamp recordings of the current response to pressure-applied NMDA/glycine in a subthalamic neuron shows that CIQ potentiation was reversible, and that NMDA-evoked currents were inhibited by D,L-APV. d. Representative whole cell voltage-clamp recording from a hippocampal CA1 pyramidal neuron in response to pressure-applied NMDA and glycine demonstrates that CIQ potentiation is selective for GluN2C/D-containing receptors over GluN2A/B-containing receptors.