Functional and Structural Interaction of (-)-Reboxetine with the Human α4β2 Nicotinic Acetylcholine Receptor

The interaction of the selective norepinephrine reuptake inhibitor, (-)-reboxetine, with the human ( h ) α4β2 nicotinic acetylcholine receptor (nAChR) in different conformational states was studied by several functional and structural approaches. Patch-clamp and Ca2+-influx results indicate that (-)-reboxetine does not activate ( h )α4β2 nAChRs via interaction with the orthosteric sites, but inhibits agonist-induced ( h )α4β2 activation by a noncompetitive mechanism. Consistently, the results from the electrophysiology-based functional approach suggest that (-)-reboxetine may act via open channel block, so it is capable of producing a use-dependent type of inhibition of the ( h )α4β2 nAChR function. We tested whether (-)-reboxetine binds to the luminal [3H]imipramine site. The results indicate that although (-)-reboxetine binds with low affinity to this site, it discriminates between the resting and desensitized ( h )α4β2 nAChR ion channels. Patch-clamp results also indicate that (-)-reboxetine progressively inhibits the ( h )α4β2 nAChR with 2-fold higher potency at the end of 1-s application of agonist compared to the peak current. The molecular docking studies show that (-)-reboxetine blocks the ion channel at the level of the imipramine locus, between M2 rings 6' and 14'. Additionally, we found a (-)-reboxetine conformer that docks within the helix bundle of the α4 subunit, near the middle region. According to molecular dynamics simulations, (-)-reboxetine binding is stable for both sites, albeit less stable than imipramine. The interaction of these drugs with the helix bundle might alter allostericaly the functionality of the channel. In conclusion, the clinical action of (-)-reboxetine may be produced (at least partially) by its inhibitory action on ( h )α4β2 nAChRs.