Abstract
To characterize the binding sites and the mechanisms of inhibition of bupropion on muscle-type nicotinic acetylcholine receptors (AChRs), structural and functional approaches were used. The results established that bupropion (a) inhibits epibatidine-induced Ca(2+) influx in embryonic muscle AChRs, (b) inhibits adult muscle AChR macroscopic currents in the resting/activatable state with approximately 100-fold higher potency compared to that in the open state, (c) increases the desensitization rate of adult muscle AChRs from the open state and impairs channel opening from the resting state, (d) inhibits binding of [(3)H]TCP and [(3)H]imipramine to the desensitized/carbamylcholine-bound Torpedo AChR with higher affinity compared to the resting/alpha-bungarotoxin-bound AChR, (e) binds to the Torpedo AChR in either state mainly by an entropy-driven process, and (f) interacts with a binding domain located between the serine (position 6') and valine (position 13') rings, by a network of van der Waals, hydrogen bond, and polar interactions. Collectively, our data indicate that bupropion first binds to the resting AChR, decreasing the probability of ion channel opening. The remnant fraction of open ion channels is subsequently decreased by accelerating the desensitization process. Bupropion interacts with a luminal binding domain shared with PCP that is located between the serine and valine rings, and this interaction is mediated mainly by an entropy-driven process.
Highlights
To characterize the binding sites and the mechanisms of inhibition of bupropion on muscle-type nicotinic acetylcholine receptors (AChRs), structural and functional approaches were used
We studied the influence of bupropion on either [3H]TCP or [3H]imipramine maximal binding to the Torpedo AChR in the resting/α-bungarotoxin (α-BTx)-bound and desensitized/CChbound states
The coinjection of epibatidine and bupropion produced an IC50 = 10.5 ± 2.1 μM (Table 1). This value is identical to that obtained by 86Rb+ efflux experiments using the same human muscle AChR [4]. Considering that this protocol is performed in the time regime of seconds, the observed blocking effect will be a combination of the effects elicited by bupropion on the open and desensitized ion channels
Summary
To characterize the binding sites and the mechanisms of inhibition of bupropion on muscle-type nicotinic acetylcholine receptors (AChRs), structural and functional approaches were used. Previous studies have shown that tricyclic antidepressants (TCAs) behave as NCAs of both muscle-type [13] and neuronal-type AChRs [14] Their noncompetitive inhibitory mechanisms on different members of the Cys-loop ligand-gated ion channel superfamily have been elucidated [13,15] [reviewed in [5]]. We want to determine the interaction of bupropion with muscle-type AChRs in different conformational states To this end, we will use binding and functional approaches including radioligand competition binding assays using well known NCAs such as [piperidyl-3, 4-3H(N)]-N-(1-(2 thienyl)cyclohexyl)-3,4-piperidine ([3H]TCP) and [3H]imipramine, Ca2+ influx and macroscopic current recordings, thermodynamic and kinetic measurements using column-immobilized Torpedo AChRs, and molecular docking and dynamics studies.
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