Allosteric inhibition of NMDA receptors by low dose ketamine

Abstract

Ketamine, a general anesthetic, has rapid and sustained antidepressant effects when administered at lower doses. Anesthetic levels of ketamine reduce excitatory transmission by binding deep into the pore of NMDA receptors where it blocks current influx. In contrast, the molecular targets responsible for antidepressant levels of ketamine remain controversial. We used electrophysiology, structure-based mutagenesis, and molecular and kinetic modeling to investigate the effects of ketamine on NMDA receptors across an extended range of concentrations. We report functional and structural evidence that, at nanomolar concentrations, ketamine interacts with membrane-accessible hydrophobic sites on NMDA receptors, which are distinct from the established pore-blocking site. These interactions stabilize receptors in pre-open states and produce an incomplete, voltage- and pH-dependent reduction in receptor gating. Notably, this allosteric inhibitory mechanism spares brief synaptic-like receptor activations and preferentially reduces currents from receptors activated tonically by ambient levels of neurotransmitters. We propose that the hydrophobic sites we describe here account for clinical effects of ketamine not shared by other NMDA receptor open-channel blockers such as memantine and represent promising targets for developing safe and effective neuroactive therapeutics.

Figure 1:

Extended dose-response relationships. (A) Two whole-cell current traces recorded from GluN1–1a/GluN2A receptors (24 cells, n >12 per concentration) expressed in HEK293 cells were elicited with glutamate (Glu, 1 mM) and −100 mV, allowed to equilibrate (ICTR), and racemic ketamine (KET) was applied at several concentrations (IKET). Panel at right shows pooled results (circles) overlayed with mono- (grey) and bi-phasic (red) dose-response functions (solid lines with associated 95% confidence intervals, shaded), and the calculated E50 values. (B) Summary of results obtained from cells co-expressing GluN1–1a with GluN2B (14 cells, n >3 cells per concentration), GluN2C (53 cells, n >10 cells per concentration), or GluN2D (37 cells, n >5 cells per concentration).

Figure 2. Putative interactions between S-KET+ and NMDA receptors.

(A) Left, results from local docking of S-KET⁺ onto an inhibited NMDA receptor conformation (6whs) illustrates three putative binding sites (boxed in left panel): one located centrally in the pore (site 1, red) and two symmetry-related lateral sites: site 2 (dark blue) and site 3 (light blue). Right, detailed positioning of S-KET⁺ in site 1 (top) and in site 2 (bottom) and predicted key contacts with residues in GluN1 (blue) and GluN2A (green) subunits. (B) Trajectories predicted from MD simulation for S-KET⁺ within the lateral sites of an inhibited (left) and an open (right) conformations (blue). (C) Putative exit pathway for S-KET⁺ from site 2 of the inhibited conformation.

Figure 3. Probing the predicted interactions between KET and NMDA receptors.

(A) Diagram highlights predicted contacts in the central and lateral sites. (B) Whole-cell traces illustrate Na⁺ currents elicited with Glu (1 mM) at pH 7.2 and −100 mV from cells expressing WT or mutated NMDA receptors, with the indicated series of KET concentrations. (C) Pooled data for mutated receptors (grey circles) (12 – 31 cells per construct, 6 – 15 cells per concentration) with overlayed best-fitting dose-response functions (solid lines with 95% confidence intervals as shaded area) and EC₅₀ values calculated with the best fitting function relative to results from WT receptors (dashed grey line).

Figure 4. Predicted contacts and effects of KET enantiomers.

(A) Detailed positioning of R-KET⁺ in site 2 of inhibited receptors illustrates its principal contacts with the receptor. (B) MD simulated trajectories for R-KET⁺ (blue) in site 2 of inhibited receptors. (C) Pooled whole-cell currents (circles) recorded from WT receptors and S-KET (left, 22 cells, n >11 per concentration) or R-KET (right, 16 cells, n >8 per concentration), overlayed with the respective best fitting dose-response functions, relative to KET (dashed line).

Figure 5:. Probing membrane-access and mechanism of receptor inhibition by low dose KET.

(A) Cartoon illustrates the setup used to record on-cell stationary Na⁺ currents from receptors isolated within the recording electrode and exposed to agonists (Glu and Gly) at pH 8, with applied +100 mV (blue). After recording basal activity for 5 min (pre), we changed conditions in the bath, and recorded activity for another 30 min (post). (B) Example traces of unitary currents recorded from WT before (pre) and after (post) equilibration in KET at the indicated concentrations (top), and the associated time-dependent change in P_o obtained after adding 0 KET (grey) and the indicated KET concentrations (red). (C) Currents recorded after adding KET (1 μM) in a pH 8 (blue) or a pH 7.2 bath (grey). (D) Activity with 10 μM KET in the bath (pH 7.2) from F636A (yellow) and WT (grey) receptors. (E) Activity from WT (left) or F636A (right) with 1 μM MEM added to a pH 7.2 (grey, and yellow) or a pH 8 bath (blue). (F) Cartoon (left) illustrates setup used to record the unitary currents (right) with KET in the recording pipette (pH 7.2). (G) Left, NMDA receptor gating scheme consisting of a top KET-free tier and a bottom KET-bound tier, with C and O representing closed-pore and open-pore states, respectively. Right, simulated current responses obtained with brief (1 ms) or sustained (5 s) Glu (1 mM) application.