Mechanism for noncompetitive inhibition by novel GluN2C/D N-methyl-D-aspartate receptor subunit-selective modulators

Abstract

The compound 4-(5-(4-bromophenyl)-3-(6-methyl-2-oxo-4-phenyl-1,2-dihydroquinolin-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)-4-oxobutanoic acid (DQP-1105) is a representative member of a new class of N-methyl-d-aspartate (NMDA) receptor antagonists. DQP-1105 inhibited GluN2C- and GluN2D-containing receptors with IC(50) values that were at least 50-fold lower than those for recombinant GluN2A-, GluN2B-, GluA1-, or GluK2-containing receptors. Inhibition was voltage-independent and could not be surmounted by increasing concentrations of either coagonist, glutamate or glycine, consistent with a noncompetitive mechanism of action. DQP-1105 inhibited single-channel currents in excised outside-out patches without significantly changing mean open time or single-channel conductance, suggesting that DQP inhibits a pregating step without changing the stability of the open pore conformation and thus channel closing rate. Evaluation of DQP-1105 inhibition of chimeric NMDA receptors identified two key residues in the lower lobe of the GluN2 agonist binding domain that control the selectivity of DQP-1105. These data suggest a mechanism for this new class of inhibitors and demonstrate that ligands can access, in a subunit-selective manner, a new site located in the lower, membrane-proximal portion of the agonist-binding domain.

Fig. 1.

Subunit selectivity of DQP-1105. A, representative current response of GluN1/GluN2D receptors expressed in X. laevis oocyte recorded during coapplication of 100 μM glutamate, 30 μM glycine (Glu/Gly), and the designated concentration of DQP-1105 (structure shown in inset). B, composite concentration-effect curves determined using two-electrode voltage-clamp electrophysiology for DQP-1105 against recombinant AMPA, kainate, and NMDA receptors expressed in X. laevis oocytes. C, concentration-effect curves determined using fluorescence-based measurements of intracellular calcium expressed as a percent of baseline fluorescence (F/F_o) for DQP-1105 inhibition of recombinant GluN1/GluN2A, GluN1/GluN2B, GluN1/GluN2C, or GluN1/GluN2D stably expressed in BHK cells (see Materials and Methods). IC₅₀ values are listed in Table 3.

Fig. 2.

DQP-1105 inhibits recombinant NMDA receptors expressed in HEK cells. A, representative traces of conventional whole cell voltage-clamp recordings of currents evoked from recombinant GluN1/GluN2A receptors by 100 μM glutamate and 100 μM glycine. Coapplication of 30 μM DQP-1105 with glutamate and glycine attenuates the steady-state current response to a new steady-state level that is 24 ± 6% (n = 5) of control. B, representative gramicidin perforated-patch, whole-cell voltage-clamp recordings from recombinant GluN1/GluN2A receptors of currents evoked by 100 μM glutamate and 100 μM glycine. DQP-1105 (30 μM) attenuated the steady-state current response to 65 ± 3.1% (n = 6) of control. C, representative perforated whole-cell voltage-clamp recordings of current responses from GluN1/GluN2D evoked by 100 μM glutamate and 100 μM glycine. DQP-1105 (30 μM) inhibited the steady-state current response upon coapplication with glutamate and glycine, with a final steady-state response of 0.2 ± 0.1% (n = 3) compared with the glutamate and glycine control. Data are from six to eight cells. D, DQP-1105 composite concentration-response curves of the steady-state current responses activated by glutamate and glycine from dialyzed whole-cell patch recordings give a fitted IC₅₀ value of 3.2 μM GluN1/GluN2D receptors and 12 μM for GluN1/GluN2A receptors (data are from six cells). E, composite concentration-response curves of the steady-state current responses to glutamate and glycine from perforated whole-cell patch recordings give an IC₅₀ of 2.1 μM GluN1/GluN2D receptors and 54 μM for GluN1/GluN2A receptors, suggesting that dialysis alters IC₅₀ of DQP-1105 at GluN1/GluN2A (data are from six to eight cells).

Fig. 3.

DQP-1105 inhibits recombinant GluN1/GluN2D receptors through a noncompetitive and voltage-independent mechanism. A, GluN1/GluN2D responses to 100 μM glutamate and 30 μM glycine were inhibited by coapplication of glutamate, glycine, and 5 μM DQP-1105, and the glutamate concentration was subsequently increased 10-fold to 1000 μM (left panel). GluN1/GluN2D responses elicited by 100 μM glutamate and 30 μM glycine were inhibited by coapplication of 5 μM DQP-1105, and the glycine concentration was subsequently increased 10-fold to 300 μM glycine (right). The increase of neither glutamate nor glycine altered the level of inhibition, suggesting a noncompetitive mechanism. B, the mean current-voltage relationship of recombinant GluN1/GluN2D receptors was determined in the absence and presence of 3–5 μM DQP-1105 (10-mV steps from −60 to +30 mV; n = 5). Error bars are S.E.M. and shown when larger than symbol size.

Fig. 4.

Determination of DQP-1105 K_D for inhibition of recombinant GluN1/GluN2D NMDA receptors expressed in HEK cells. A, conventional whole cell voltage-clamp recordings of currents evoked from recombinant GluN1/GluN2D receptors by 2-s applications of 1000 μM NMDA and 50 μM glycine. Coapplication of DQP-1105 with NMDA and glycine caused a concentration-dependent attenuation of the steady-state current response, with a final steady-state response of 3.4 ± 1.1% (n = 8) at 30 μM DQP-1105 compared with control. B, the composite concentration-response relationship of the steady-state current responses to 1000 μM NMDA plus 30 μM glycine shows that DQP-1105 inhibits GluN1/GluN2D receptors with an IC₅₀ value of 1.9 μM. C, there is a linear relationship between 1/τ_ONSET and DQP-1105 concentration, suggesting that the time-dependent inhibition of NMDA-activated GluN1/GluN2D reflects the association of DQP-1105 with its binding site. From this linear relationship, K_D can be calculated from k_ON and k_OFF, and was 1.4 μM for GluN1/GluN2D receptors, similar to the measured IC₅₀ value.

Fig. 5.

Inhibition by DQP-1105 is dependent upon binding of NMDA to the GluN2D subunit. Current responses were evoked by agonist application to HEK cells transiently expressing GluN1/GluN2D receptors that were recorded under voltage clamp at −60 mV. A, GluN1/GluN2D receptors were activated by a 5-s application of 200 μM NMDA in the continuous presence of 100 μM glycine and subsequently were activated by 5-s applications of 200 μM NMDA in the continuous presence of both 100 μM glycine and 3 μM DQP-1105. These recordings displayed a pronounced relaxation of the current response after NMDA binding, with an 84 ± 1.9% peak response compared with control and a steady-state response of 21 ± 1.6% compared with control (n = 7). B, GluN1/GluN2D receptors were activated by a 5-s application of 300 μM glycine in the continuous presence of 20 μM NMDA, and subsequently activated by a 5-s application of 300 μM glycine in the continuous presence of both 20 μM NMDA and 3 μM DQP-1105. These recordings did not show a prominent relaxation of response, with a peak response of 29 ± 0.9% compared with control and a steady-state response of 24 ± 1.6% of the control response amplitude (n = 5). C, although the peak responses of GluN1/GluN2D receptors activated by NMDA or glycine after pretreatment with 3 μM DQP-1105 were significantly different (p < 0.05; paired t test), the steady-state responses (D) were not significantly different (paired t test). These data suggest that DQP-1105 shows higher potency after NMDA (but not glycine) binding to the receptor.

Fig. 6.

DQP-1105 increases mean shut time and decreases open probability of GluN1/GluN2D receptors. A, unitary currents recorded from outside-out patches from HEK cells expressing GluN1/GluN2D were activated by glutamate (1 mM) and glycine (50 μM) at −80 mV B, after a 2-min recording in control conditions, 3 μM DQP-1105 was coapplied with agonists to the patch and currents were recorded for 2 min. C, 30 μM DQP-1105 was then coapplied to the same patch and currents recorded for 2 min. For A to C, the boxed region in the top trace is shown on an expanded time scale in the three lower traces. Individual openings and closings were idealized by time course fitting (see Materials and Methods) and composite open duration (D) and shut duration (E) histograms were constructed from idealized data from four patches. The open-period distribution was fitted with the sum of two exponential components (Table 4) and the shut time distribution was fitted with the sum of five exponential components with τ (percentage area) of 0.026 (27%), 0.16 (11%), 1.4 (9%), 7.2 (43%), and 22 ms (10%) for control (7547 intervals), 0.016 (36%), 0.13 (12%), 3.3 (9%), 14 (34%), and 55 ms (9%) for 3 μM DQP-1105 (5617 intervals), and 0.026 (27%), 0.23 (13%), 11 (22%), 85 ms (18%), and 1500 (20%) for 30 μM DQP-1105 (952 intervals).

Fig. 7.

Identification of structural determinants of GluN2D-selective inhibition using chimeric GluN2A-GluN2D receptors. A, indicated regions of the polypeptide were exchanged between wild-type GluN2A and GluN2D. Concentration-effect curves at GluN1/GluN2D, GluN1/GluN2A, GluN1/GluN2A(2D-S1S2), GluN1/GluN2A(2D-S1), and GluN1/GluN2A(2D-S2), B, GluN1/GluN2A(2D-M1M2M3), GluN1/GluN2A(2D-ATD), and GluN1/GluN2A(ΔATD) receptors were analyzed, and the mean IC₅₀ values (± S.E.M.) are shown. These data suggest that the S2 region transfers DQP-1105 sensitivity to GluN2A. C, linear representation of the S2 regions of GluN2A(2D-S2) in which regions of GluN2D-S2 have been reverted to that of GluN2A to probe for loss of inhibition of DQP-1105. D, the inhibition produced by 5 μM DQP-1105 in the presence of 1000 μM glutamate and 300 μM glycine is shown as a percent of control. Three chimeric receptors show significantly reduced inhibition compared with GuN2A(S2) chimera (p < 0.05, one-way ANOVA with Tukey's test, n = 5–13 oocytes per chimeric receptor). E, site-directed mutagenesis of residues within GluN2D S2 chimeric regions identified Gln701 and Leu705 (asterisks in B) as key structural determinants for antagonist activity of DQP-1105 at GluN2D receptors (p < 0.05, one-way ANOVA with Tukey's test; n = 4–11 oocytes per mutant receptor).

Fig. 8.

Homology models of GluN1-GluN2 receptors shows that residues identified in lower lobe of the clamshell shaped ligand binding domain are localized near each other in three-dimensional space and in close proximity to the linkers that connect the ligand binding domain to the transmembrane helices. A, left, a GluN1-GluN2D receptor homology model (ATD omitted); right, shows a GluN1-GluN2A homology model (ATD omitted). B, the GluN2D-subunit is expanded (left) to show the lower portion of the ligand-binding domain containing residues Gln701 and Leu705 (red), which are critical divergent structural determinants for the antagonist activity of DQP-1105. The S1-M1 and M3-S2 linker regions are also shown in red. A GluN2A-subunit is expanded (right) to show the same region with the residues corresponding to GluN2D-Gln701(Tyr in GluN2A, yellow) and GluN2D-Leu705 (Phe in GluN2A yellow); linkers are also shown in yellow. Compound DQP-1105 is shown in the lower panel on the same scale.