Different interaction between tricyclic antidepressants and mecamylamine with the human α3β4 nicotinic acetylcholine receptor ion channel

The interaction of tricyclic antidepressants (TCAs) with the human (h)α3β4 nicotinic acetylcholine receptor (AChR) in different conformational states was compared with that for mecamylamine by using functional and structural approaches including, Ca²⁺ influx, radioligand binding, and molecular docking. The results established that: (a) [³H]imipramine binds to a single site with relatively high affinity (K_d = 0.41 ± 0.04 μM), (b) imipramine inhibits [³H]imipramine binding to the resting/κ-bungarotoxin-bound AChR (K_i = 0.68 ± 0.08 μM) with practically the same affinity as to the desensitized/epibatidine-bound AChR (K_i = 0.83 ± 0.08 μM), suggesting that TCAs do not discriminate between these conformational states, and (c) although TCAs (IC₅₀ ∼1.8-2.7 μM) and mecamylamine (IC₅₀ = 3.3 ± 0.4 μM) inhibit (±)-epibatidine-induced Ca²⁺ influx with potencies in the same concentration range, TCAs (K_i ∼1-3.6 μM), but not mecamylamine (apparent IC₅₀ ∼0.2 mM), inhibit [³H]imipramine binding to hα3β4 AChRs in different conformational states. This is explained by our docking results where imipramine, in the neutral and protonated states, interacts with the leucine (position 9′) and valine/phenylalanine (position 13′) rings, whereas protonated mecamylamine (>99% at physiological pH) interacts with the outer ring (position 20′). Our data indicate that TCAs bind to overlapping sites located between the serine and valine/phenylalanine rings in the hα3β4 AChR ion channel, whereas protonated mecamylamine can be attracted to the channel mouth before blocking ion flux by interacting with a luminal site in its neutral state.