Allosteric Site Mediates Inhibition of Tonic NMDA Receptor Activity by Low Dose Ketamine

Abstract

Ketamine, a general anesthetic, has rapid and sustained antidepressant effects when administered at lower doses. At anesthetic doses, ketamine causes a drastic reduction in excitatory transmission by lodging in the centrally located hydrophilic pore of the NMDA receptor, where it blocks ionic flow. In contrast, the molecular and cellular targets responsible for the antidepressant effects of ketamine remain controversial. Here, we report functional and structural evidence that, at nanomolar concentrations, ketamine interacts with membrane-accessible hydrophobic sites where it stabilizes desensitized receptors to cause an incomplete, voltage- and pH-dependent reduction in NMDA receptor activity. This allosteric mechanism spares brief receptor activations and reduces preferentially currents from tonically active receptors. The hydrophobic site is a promising target for safe and effective therapies against acute and chronic neurodegeneration.

Figure 1. Extended Dose Response Relationship.

(A) Whole-cell current traces elicited with glutamate (Glu) and with two series of ketamine (KET) concentrations, from GluN1-1a/GluN2A (WT) receptors expressed in HEK293 cells, are shown normalized to the control steady-state current level (I_CTR). (B) Summary of pooled results (black circles, n = 24 cells, n >12 cells per concentration) and fitted mono- (blue) and bi-phasic (grey) dose-response functions (solid lines) with associated 95% confidence intervals (shaded). Indicated F and p statistics were calculated as described in Methods.

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

(A) Left, results from local docking of protonated S(−)-ketamine onto an inactive NMDA receptor structure lacking C-terminal domains (PDB ID: 6whs) illustrates three putative binding sites (boxed in left panel): one located centrally in the pore (site 1, red) and two symmetry-related intra-membrane 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 key contacts with residues in GluN1 (blue) and GluN2A (green) subunits. (B) Results from MD simulation of S-KET+ with inactive (left) and active (right)

Figure 3. Probing putative contacts between KET and NMDA receptors.

(A) Whole-cell 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. (B) Extended dose response relationships illustrated as best fitting functions (solid lines) with associated 95% confidence intervals (shades area) to pooled data for each receptor (n, 12 – 31 cells per construct, 6 – 15 cells per concentration). (C) Results from MD simulation of protonated R(+)-ketamine onto an inactive receptor conformation illustrates principal contacts with residues in site 2. (D) Results from MD simulation of R-KET+ with inactive NMDA receptor structures illustrates its trajectories (blue). (E) Extended dose-response relationships for WT receptors with ketamine isoforms are illustrated as best fits of biphasic models (lines) and 95% confidence intervals (shaded area) of pooled results from whole-cell Na+ currents recorded at pH 7.2 and −100 mV for each ketamine preparation. n = 8 – 11 cells per enantiomer, 4 – 11 cells per concentration.

Figure 4. Probing a trans-membrane pathway for drug access to effector sites within NMDA receptors.

(A) Cartoon illustrates setup used to record on-cell stationary Na⁺ currents from NMDA receptors isolated within the recording electrode containing agonists (Glu and Gly) at pH 8 (blue). After recording basal activity (pre, unshaded) for 5 min, KET (or MEM) is added to the bath at pH 7.2 (grey) or as indicated, and activity is recorded for another 30 min (post, shaded). (B) Top, Currents recorded from WT receptors before (pre) and after equilibration in KET at the indicated concentrations (post). Bottom, time-dependent change in calculated Po along individual recordings obtained with 0 KET (grey) and the indicated KET concentrations (red). (C) Currents recorded after adding KET (1 μM) in the bath at pH 8 (blue) relative to pH 7.2 (grey). (D) Activity from GluN1/GluN2AF636A (yellow) relative to WT (grey) with 10 μM KET. (E) Activity from WT with 1 μM MEM added to the bath at pH 7.2 (grey) relative to pH 8 (blue). (F) Activity from GluN1/GluN2AF636A (yellow) relative to WT (grey) with 1 μM MEM in the bath at pH 7.2.

Figure 5. Probing the inhibitory mechanism of low-dose ketamine.

(A) Cartoon of the setup used to record on-cell stationary Na⁺ currents from NMDA receptors isolated within a membrane patch exposed to agonists (Glu and Gly) and KET, at pH 7.2, and +100 mV. KET can access its binding sites through the open pore, together with Na⁺, and through the pipette-enclosed membrane-patch. (B) Inward Na⁺ currents recorded from individual receptors with the indicated sub-micromolar concentrations of KET. (C) Kinetic model used to fit the sequence of closed and open intervals observed in single-channel recordings includes gating transitions among agonist-bound closed (C1-C5) and open (O) kinetic states that are KET-free (top tier) and KET-bound (bottom tier), respectively. Two glutamate-binding steps initiate the activation of resting (C₀₀ and C₀) receptors, and a KET-binding step connects the two tiers. Six hypothetical models (M_C1-M_C5, and M_B) differ in the kinetic state (C₁-C₅, or O) proposed to allow transition between tiers with highest probability. (D) Schematic of a three-pulse stimulation protocol illustrates the timing and duration of consecutive Glu-KET-Glu applications. Top traces (thin lines) represent the macroscopic current simulated with model M_C5. Bottom traces (thick lines) illustrate whole-cell currents recorded experimentally. (E) Summary of results illustrates the increase in current response to the second glutamate pulse (I_Dt) with time (Dt), as predicted (colored lines) with the models in panel D, and as measured experimentally (circles) with fitted exponential function (grey line).