Perszyk RE et al. (2020), Biased modulators of NMDA receptors control cha...

XB-ART-56935

Nat Chem Biol 2020 Feb 01;162:188-196. doi: 10.1038/s41589-019-0449-5.

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Biased modulators of NMDA receptors control channel opening and ion selectivity.

Perszyk RE, Swanger SA, Shelley C, Khatri A, Fernandez-Cuervo G, Epplin MP, Zhang J, Le P, Bülow P, Garnier-Amblard E, Gangireddy PKR, Bassell GJ, Yuan H, Menaldino DS, Liotta DC, Liebeskind LS, Traynelis SF.

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Allosteric modulators of ion channels typically alter the transitions rates between conformational states without changing the properties of the open pore. Here we describe a new class of positive allosteric modulators of N-methyl D-aspartate receptors (NMDARs) that mediate a calcium-permeable component of glutamatergic synaptic transmission and play essential roles in learning, memory and cognition, as well as neurological disease. EU1622-14 increases agonist potency and channel-open probability, slows receptor deactivation and decreases both single-channel conductance and calcium permeability. The unique functional selectivity of this chemical probe reveals a mechanism for enhancing NMDAR function while limiting excess calcium influx, and shows that allosteric modulators can act as biased modulators of ion-channel permeation.

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GO keywords: ion channel activity

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	Figure 2. EU1622-1 reduces single channel conductance for GluN1/GluN2B NMDARs. (A) Unitary currents were recorded from an outside-out patch excised from an HEK cell co-transfected with GluN1, GluN2B, and GFP. The holding potential was âˆ’80 mV and channel openings are shown as downward by convention. Responses were produced by the application of external solution with 0.1 mM glutamate plus 0.05 mM glycine (0.5 mM Ca2+). NMDARs showed reduced unitary current amplitude when 50 Î¼M EU1622-1 was co-applied with the agonist (lower panel) compared to vehicle (upper panel). (C) Composite amplitude histograms determined by time course fitting were analyzed by fitting with the sum of multiple Gaussian components by maximum likelihood for vehicle (open bars, solid line, left y-axis) and 30â€“50 Î¼M EU1622-1 (filled bars, dashed line, right y-axis). Supplementary Table 8 provides a summary of chord conductance. (D) Composite open duration histograms for the patch shown and fitted by the sum of two exponential components displayed similarly to (C). Mean open time is given in Supplementary Table 8; individual fitted time constants Â± standard deviation and area for the fits were: vehicle Tau1 0.10 Â± 0.0028 ms, 12%, Tau2 2.3 Â± 0.31 ms, 88%, EU1622-1 Tau1 0.54 Â± 0.40 ms, 26%, Tau2 3.2 Â± 0.93 ms, 74% (n = 3 patches). (E) Examples of direct sublevel transitions in the presence of EU1622-1 are shown, marked by arrowheads.
	Figure 3. EU1622-14 prolongs the response deactivation time course and reduces channel conductance of NMDARs. (A) Representative GluN1/GluN2B NMDAR current responses during rapid application of 1/0.1 mM glutamate/glycine for 10 ms (triangle) in 100 Î¼M glycine (white bar) with vehicle or EU1622-14 (30 Î¼M, Compound 2). Horizontal triangles show peak amplitudes. An expanded, normalized view is shown on the right to highlight the deactivation time course of the responses. (B) Peak amplitude (left) and Ï„weighted of the deactivation (right) of rapid glutamate applications (10 ms) with and without EU1622-14 for all diheteromeric NMDARs (see Supplementary Table 4). (C) Representative slow perfusion stimulated GluN1/GluN2B responses with glutamate and glycine co-applied with vehicle or E1622-14. The NMDAR-mediated increase in variance can be seen from the high-pass filtered current (grey, below). (D) Current-variance plot of the rise and decay of the low concentration agonist application from (C). (E) Mean conductance of all diheterometic NMDARs with and without EU1622-14 (50 Î¼M). For summary data see Supplementary Table 4. For all panels, * p<0.05 by paired t-test, with the Holm-Bonferroni correction to control for the family wise error rate in (B).
	Figure 4. EU1622-14 reduces native NMDAR conductance. Unitary currents activated by 500 μM NMDA and 100 μM glycine (−80 mV) in outside-out patches were from cortical neurons in 1.25 (A) or 0.5 mM Ca2+ (B); scale bars are 2 s (A1), 100 ms (A2) and 50 ms (A3). The dashed line is 5 pA. “C” indicates closed state and arrowheads in A3 show sublevel transitions in EU1622-14. Vehicle was 0.25% DMSO. (C,D) Amplitudes determined (6,369 vehicle, 14,406 EU1622-14) from 6 patches in 1.25 mM Ca2+ or 4 patches (3,473 vehicle, 6,639 EU1622-14) in 0.5 mM Ca2+ were fitted to the sum of 2–3 Gaussians (smooth line is the probability density function). The amplitude ± standard deviation and area for the fits (1.25 mM Ca2+) were: vehicle Amp1 −4.13 ± 0.36 pA, 85%, Amp2 −3.52 ± 0.83 pA, 15%, EU1622-14 Amp1 −3.45 ± 0.59 pA, 71% Amp2 −2.58 ± 0.22 pA, 23% Amp3 −2.16 ± 0.57 pA, 6 %. The amplitude ± standard deviation and area for the fits (0.5 mM Ca2+) were: vehicle Amp1 −4.79 ± 0.35 pA, 73% Amp2 −4.16 ± 0.75 pA, 27%, EU1622-14 Amp1 −4.23 ± 0.55 pA, 79%, Amp2 −3.17 ± 0.26 pA, 16% Amp3 −2.27 ± 0.65 pA, 4%.
	Figure 5. EU1622-14 reduces Ca2+ permeation through NMDARs. (A) Representative I-V curves for GluN1/GluN2A responses to maximally-effective agonist co-applied with vehicle (left) or EU1622-14 (right) in the low/high Ca2+. A high monovalent method was used to determine the relative Ca2+/monovalent permeability ratio. The fitted curve is a fourth-order polynomial. (B) The average reversal potentials are given for GluN1/GluN2A and GluN1/GluN2B (vehicle vs. EU1622-14). (C) The mean ΔReversal potentials (high Ca2+ minus low Ca2+) are given. (D) The average Ca2+ permeability ratio to monovalent ions is from the Lewis equation. *p<0.05 (One-way ANOVA, post-hoc t-test of drug vs vehicle, Bonferroni correction for multiple comparisons); F3,28 = 6.91. For summary data see Supplementary Table 9.
	Figure 6. EU1622-14 biasedly modulates Na+ and Ca2+ influx through NMDARs. (A-C) Simultaneous patch-clamp/imaging recordings from cortical neuronal cultures illustrate a significant augmentation of total current with a non-significant increase in the Ca2+ fluorescence (stimulated by 500/100 Î¼M NMDA/glycine) due to EU1622-14 modulation. (A) Time-lapse imaging of a Ca2+-sensitive, cell impermeant dye (Calbryte 590 potassium-salt, images were taken at a 1s interval). Representative vehicle and EU1622-14 fluorescence B, left) and current (C, left) responses to 500/100 Î¼M NMDA/glycine co-applied with vehicle (equivalent volume DMSO) or EU1622-14 (50 Î¼M) following a 1 min pre-application of vehicle or drug. Summary graphs are shown on the right of each figure panel. A significant increase in the peak current amplitude (p = 0.0004, unpaired t-test) was detected in the presence of EU1622-14 (50 Î¼M) but not a significant increase in the slope of the Ca2+-fluorescence signal (p = 0.09, unpaired t-test). (D-G) Simultaneous fluorescence monitoring of Na+ and Ca2+ influx using specific cell permeable dyes illustrates selective modulation by EU1622-14. (D) Intracellular Na+ and Ca2+ levels were measured in cortical neuronal cultures utilizing time-lapse imaging of Na+- and Ca2+-sensitive dyes. 500/100 Î¼M NMDA/glycine were co-applied in vehicle (0.5% DMSO) or EU1622-14 (50 Î¼M) following a 1 min pre-application of vehicle or drug. Images were acquired every 4 s. Boxed regions in (E) are shown in (D). (F-G) Cellular fluorescence responses to NMDAR stimulation in the presence of vehicle or EU1622-14. Agonist solution perfusion (indicate by the arrow, 500/100 Î¼M NMDA/glycine, with vehicle or EU1622-14) resulted in a rise in the CoroNa AM dye (F, Na+ response) and the Calbryte 590 AM dye (G, Ca2+ response). For each trial, the slope of the rise of the respective signals were measured for all neurons in the camera view, then averaged to obtain one experimental data point (shown in the plot on the right). Scale bars denote 50 Î¼m.

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