The Negative Allosteric Modulator EU1794-4 Reduces Single-Channel Conductance and Ca2+ Permeability of GluN1/GluN2A N-Methyl-d-Aspartate Receptors

Abstract

NMDA receptors are ligand-gated ion channels that mediate a slow, Ca²⁺-permeable component of excitatory synaptic currents. These receptors are involved in several important brain functions, including learning and memory, and have also been implicated in neuropathological conditions and acute central nervous system injury, which has driven therapeutic interest in their modulation. The EU1794 series of positive and negative allosteric modulators of NMDA receptors has structural determinants of action near the preM1 helix that is involved in channel gating. Here, we describe the effects of the negative allosteric modulator EU1794-4 on GluN1/GluN2A channels studied in excised outside-out patches. Coapplication of EU1794-4 with a maximally effective concentration of glutamate and glycine increases the fraction of time the channel is open by nearly 1.5-fold, yet reduces single-channel conductance by increasing access of the channel to several subconductance levels, which has the net overall effect of reducing the macroscopic current. The lack of voltage-dependence of negative modulation suggests this is unrelated to a channel block mechanism. As seen with other NMDA receptor modulators that reduce channel conductance, EU1794-4 also reduces the Ca²⁺ permeability relative to monovalent cations of GluN1/GluN2A receptors. We conclude that EU1794-4 is a prototype for a new class of NMDA receptor negative allosteric modulators that reduce both the overall current that flows after receptor activation and the flux of Ca²⁺ ion relative to monovalent cations. SIGNIFICANCE STATEMENT: NMDA receptors are implicated in many neurological conditions but are challenging to target given their ubiquitous expression. Several newly identified properties of the negative allosteric modulator EU1794-4, including reducing Ca²⁺ flux through NMDA receptors and attenuating channel conductance, explain why this modulator reduces but does not eliminate NMDA receptor function. A modulator with these properties could have therapeutic advantages for indications in which attenuation of NMDA receptor function is beneficial, such as neurodegenerative disease and acute injury.

Graphical abstract

Fig. 1.

EU1794-4 is a NMDAR negative allosteric modulator with submaximal inhibition. (A) Representative EU1794-4 concentration-response recordings from X. laevis oocytes expressing the diheteromeric NMDARs activated by glutamate and glycine (100 and 30 μM). Increasing concentrations (0.1, 0.3, 1, 3, 10, and 30 μM) of EU1794-4 are illustrated by the shaded boxes above each recording. (B) Mean ± S.E.M. EU1794-4 concentration-response data are shown; smooth line is a least-squares fit of the data by the Hill equation. Each concentration-response curve was fit individually by the Hill equation. For GluN1/GluN2A (n = 9), the IC₅₀ [mean (95% confidence interval determined from logIC₅₀)] was 3.0 (2.0, 4.7) μM with a residual response in saturating concentration of EU1794-4 (as percentage of control) of 27% ± 3% and a Hill slope of 1.03 ± 0.12. For GluN1/GluN2B (n = 12), the IC₅₀ was 3.0 (0.79, 11) μM with a residual response of 53% ± 6% and a Hill slope of 0.46 ± 0.05. For GluN1/GluN2C (n = 13), the IC₅₀ was 0.35 (0.30, 0.40) μM with a residual response of 47% ± 2% and a Hill slope of 0.89 ± 0.03. For GluN1/GluN2D (n = 8), the IC₅₀ was 0.28 (0.23, 0.33) μM with a residual response of 55% ± 2% and a Hill slope of 1.1 ± 0.02. The scale bar depicts 100 μA and 60 seconds. (C) The chemical structure of EU1794-4 indicating the potentially ionizable nitrogens, with pKa values estimated by using Jaguar pKa implemented in Schrödinger Release 2020-4 (Klicić et al., 2002; Bochevarov et al., 2016; Yu et al., 2018). Considering that tautomers may occur (Attanasi et al., 2008; Josey et al., 2019), we calculated the pKas for all ionizable groups in both tautomers. The percent of compound ionized was calculated from the Henderson-Hasselbalch equation assuming each tautomer is pure.

Fig. 2.

Single-channel recording from a HEK-293 cell expressing GluN1/GluN2A. (A) Representative unitary currents (filtered at 1 kHz for display) recorded from an outside-out patch expressing GluN1/GluN2A (A₁) in control (top) or in the presence of EU1794-4 (bottom). A portion, illustrated by the box in A₁, of the same recording on an expanded time scale are shown in (A₂). (B) All-point response amplitude histograms of the patch recording in (A) (of the single-channel range) reveals lower conductance levels in the presence of EU1794-4, including a brief state that overlaps with the baseline patch noise. The control histogram was fitted by three Gaussian distributions and the EU1794-4 histogram was fitted by four Gaussian distributions.

Fig. 3.

EU1794-4 alters several parameters of GluN1/GluN2A receptor channel gating. (A) Open state duration histograms for control (top) and EU1794-4 (bottom) fitted by the sum of two to three exponential components (individual components are shown with dotted lines). Open durations were determined as the duration of each open period regardless of opening conductance level. (B) Closed state duration histograms for control (top) and EU1794-4 (bottom) were fitted by the sum of five exponential components (individual components are shown with dotted lines). The histogram are plotted using the square root (SQRT) of the counts.

Fig. 4.

Direct transitions between EU1794-4 conductance states are primarily between adjacent levels. (A) The amplitude histograms from time course fitting from the same patch as shown in Fig. 1 are given for control (left) and EU1794-4 (right), fitted by the sum of two to three Gaussian components (individual components are shown with dotted lines). (B) Examples of direct transitions between the 60 and 47 pS states (top) and between the 47 and 23 pS states (bottom) are shown. (C) A scatterplot of the contiguous openings with direct transitions between the channel sublevels (only durations greater than 2.5 filter rise times were included) for the patch shown in Fig. 2 (control, left; EU1794-4, right). For each transition, the initial amplitude is on the x-axis and the final amplitude on the y-axis. The A_CRIT values (broken lines) were calculated from the amplitude histogram fitting in (A). The relative proportion of all direct transitions (determined by A_CRIT) is given for all possible transitions between sublevels in all patches. When multiple direct transitions between subconductance levels occurred, only the first two were analyzed.

Fig. 5.

EU1794-4 reduces weighted mean conductance in NMDARs. (A) Example GluN1/GluN2A current responses to slow perfusion of glutamate with constant exposure to glycine supplemented with vehicle or EU1794-4. The increase in variance of the NMDAR response (black trace, below) is apparent in the 1 Hz high-pass filtered current (gray trace, above). (B) Current-variance relationship determined from the onset and washout of the current response to low concentration of agonist from (A). The smooth line is the fit of the current-variance equation to the data (see Materials and Methods). (C) Weighted mean conductance is given for all diheteromeric NMDARs with and without EU1794-4 (30 µM). Conductance was 55 ± 2.8 pS (control) and 39 ± 2.2 pS (EU1794-4) for GluN1/GluN2A, 50 ± 1.2 pS (control) and 31 ± 0.86 pS (EU1794-4) for GluN1/GluN2B, 19 ± 2.8 pS (control) and 15 ± 2.5 pS (EU1794-4) for GluN1/GluN2C, and 28 ± 1.4 pS (control) and 28 ± 2.5 pS (EU1794-4) for GluN1/GluN2D. *P < 0.05 by paired t test.

Fig. 6.

EU1794-4 reduces the relative Ca²⁺ permeability of GluN1/GluN2A receptors. (A) A representative current-voltage relationship for GluN1/GluN2A current responses activated by maximally effective agonist coapplied with vehicle (left) or EU1794-4 (right) when extracellular solutions contained low or high concentration of Ca²⁺. The reversal potentials (determined by least-squares fitting using a fourth-order polynomial) were used with the Lewis equation (see Materials and Methods) to determine the relative Ca²⁺/monovalent permeability ratio. (B) The average reversal potentials are shown for vehicle and EU1794-4. (C) The mean ΔReversal potentials (high Ca²⁺ minus low Ca²⁺) are given. (D) The average Ca²⁺ permeability ratio to monovalent ions was calculated from the Lewis equation. *P < 0.05 (unpaired t test).