Enantioselective interactions of dextromethorphan and levomethorphan with the α3β4-nicotinic acetylcholine receptor: comparison of chromatographic and functional data

The enantioselectivity of the interaction of dextromethorphan (DM) and levomethorphan (LM) with an immobilized α3β4 subtype of the nicotinic acetylcholine receptor (nAChR) liquid chromatographic stationary phase has been compared to DM- and LM-induced non-competitive blockade of nicotine-stimulated 86Rb+ efflux from cells expressing the α3β4-nAChR. The association rate constants (kon) and dissociation rate constants (koff) for the formation of the DM and LM complexes with the nAChR were determined using non-linear chromatographic techniques and the koff value for DM (1.01±0.01 s−1) was significantly lower than the koff for LM (1.55±0.002 s−1) while the kon values did not significantly differ (23.66±0.61 and 18.61±0.36 μM−1 s−1, respectively). In thermodynamic studies using the van’t Hoff approach, the enthalpy change (ΔH°) of the DM–nAChR complex was 330 cal mol−1 more stable than the LM–nAChR complex, while there was no significant difference in the entropy change (ΔS°). In the functional in vitro cell-based studies, there was no significant difference in the observed IC50 values for DM (10.1±1.01 μM) and LM (10.9±1.08 μM), but the recovery from the DM-induced blockade was slower than the recovery from LM-induced blockade; after 7 min: 38.25±15.46% recovery from DM blockade, 63.30±16.08% from LM blockade; after 4 h: 76.20±4.51% recovery from DM blockade and 93.12±8.76% from LM blockade. The enantioselective differences in the functional effects are consistent with the chromatographic and thermodynamic data and indicate that this difference is due to increased stability of the DM–nAChR complex. The results suggest that the chromatographic approach can be used to probe the interaction of non-competitive inhibitors (NCIs) with nAChRs and to predict relative duration of functional blockades.