Sub-anesthetic concentrations of (R,S)-ketamine metabolites inhibit acetylcholine-evoked currents in α7 nicotinic acetylcholine receptors

Abstract

The effect of the (R,S)-ketamine metabolites (R,S)-norketamine, (R,S)-dehydronorketamine, (2S,6S)-hydroxynorketamine and (2R,6R)-hydroxynorketamine on the activity of α7 and α3β4 neuronal nicotinic acetylcholine receptors was investigated using patch-clamp techniques. The data indicated that (R,S)-dehydronorketamine inhibited acetylcholine-evoked currents in α7-nicotinic acetylcholine receptor, IC(50) = 55 ± 6 nM, and that (2S,6S)-hydroxynorketamine, (2R,6R)-hydroxynorketamine and (R,S)-norketamine also inhibited α7-nicotinic acetylcholine receptor function at concentrations ≤ 1 μM, while (R,S)-ketamine was inactive at these concentrations. The inhibitory effect of (R,S)-dehydronorketamine was voltage-independent and the compound did not competitively displace selective α7-nicotinic acetylcholine receptor ligands [(125)I]-α-bungarotoxin and [(3)H]-epibatidine indicating that (R,S)-dehydronorketamine is a negative allosteric modulator of the α7-nicotinic acetylcholine receptor. (R,S)-Ketamine and (R,S)-norketamine inhibited (S)-nicotine-induced whole-cell currents in cells expressing α3β4-nicotinic acetylcholine receptor, IC(50) 3.1 and 9.1 μM, respectively, while (R,S)-dehydronorketamine, (2S,6S)-hydroxynorketamine and (2R,6R)-hydroxynorketamine were weak inhibitors, IC(50) >100 μM. The binding affinities of (R,S)-dehydronorketamine, (2S,6S)-hydroxynorketamine and (2R,6R)-hydroxynorketamine at the NMDA receptor were also determined using rat brain membranes and the selective NMDA receptor antagonist [(3)H]-MK-801. The calculated K(i) values were 38.95 μM for (S)-dehydronorketamine, 21.19 μM for (2S,6S)-hydroxynorketamine and>100 μM for (2R,6R)-hydroxynorketamine. The results suggest that the inhibitory activity of ketamine metabolites at the α7-nicotinic acetylcholine receptor may contribute to the clinical effect of the drug.

Figure 1

The structures of the compounds used in this study

Figure 2

The effect of 10, 50 and 200nM (R,S)-dehydronorketamine {(R,S)-DHNK} on the current traces recorded using KXα7R1 cells in the whole-cell configuration of patch-clamp technique in the presence of 1 mM ACh with a holding potential of −80mV.

Figure 3

A. The effect of 30 nM (R,S)-DHNK on the ACh-induced current in KXα7R1 cells illustrates a reversible pronounced (45%) inhibition of the whole-cell current. Holding potential was −80 mV. B. Dose-dependent effect of (R,S)-DHNK on the ACh-induced current in KXα7R1 cells. The curve was fitted to Hill equation. Symbols and bars represent the mean ± S.E.M.

Figure 4

A. Superimposed traces of the ACh-induced currents in the absence and presence of DHNK in KXα7R1 cells. B. Voltage-dependence of inhibitory effect of (R,S)-DHNK on the ACh-induced current in KXα7R1 evoked at various holding potentials from −100 to +80 mV, quantified at its maximal amplitude in the absence (circles) and presence (triangles) of 30 nM DHNK and plotted versus the corresponding holding potential.

Figure 5

Competitive displacement of [¹²⁵I]-α-BTX and [³H]-EB from cellular membranes obtained from the KXα7R1 cell line by (R)-DHNK, (S)-DHNK, MLA and (S)-nicotine.

Figure 6

The effect of the concentration of (R,S)-ketamine {(R,S)-Ket}, (R,S)-norketamine {(R,S)-norKet}, (2S,6S)-hydroxynorketamine {SS-HNK}, (2R,6R)-hydroxynorketamine {RR-HNK} and (R,S)-dehydronorketamine {(R,S)-DHNK} on the (S)-nicotine-induced current in KXα3β4R2 cells determined using whole-cell configuration of the patch-clamp technique. The calculated IC₅₀ values are included in the figure.