Interaction of benzylidene-anabaseine analogues with agonist and allosteric sites on muscle nicotinic acetylcholine receptors

Background and purpose: Benzylidene-anabaseines (BAs) are partial agonists of the α7 nicotinic acetylcholine receptor (nAChR) but their mechanism(s) of action are unknown. Our study explores several possibilities, including direct interactions of BAs with the nAChR channel. Experimental approach: Functional and radioligand-binding assays were used to examine the interaction of two BA analogues, 3-(2,4-dimethoxybenzylidene)-anabaseine (DMXBA) and Its primary metabolite 3-(4-hydroxy-2-methoxybenzylidene)anabaseine (4OH-DMXBA) with both agonist and non-competitive antagonist (NCA)-bindlng sites on muscle-type nAChRs. Key results: Both BAs non-competitively inhibited ACh activation of human fetal muscle nAChRs and sterlcally inhibited the specific binding of the NCAs [piperidyl-3,4-3H(N)]-(N-(1 -(2-thienyl)cyclohexyl)-3,4-plperidine ([ ³H]TCP) and [ ³H]dizocilpine to Torpedo nAChRs in the desensitized state. These compounds modulated [3H]tetracaine, [ ¹⁴C]amobarbital and [ ³H]TCP binding to resting nAChRs by allosterlc mechanisms. Both BAs enhanced [ ³H]TCP binding when the nAChR was initially in the resting but activatable state, suggesting that both compounds desensitized the Torpedo nAChR. Although DMXBA failed to activate human fetal muscle nAChRs, 40H-DMXBA was found to be a partial agonist. [ ³H]Nlcotine competition-binding experiments confirmed that 40H-DMXBA has higher affinity than DMXBA for the agonist sites, and that DMXBA Is also a competitive antagonist. Conclusions and implications: 3-(4-hydroxy-2- methoxybenzylidene)-anabaseine is a partial agonist for human fetal muscle nAChRs, whereas DMXBA only has competitive and NCA activities. The NCA-bindlng site for BAs overlaps both the phencyclidine- and dizocilpine-binding sites in the desensitized Torpedo nAChR ion channel. The desensitizing property of BAs suggests another possible mode of non-competitive inhibition in addition to direct channel-blocking mechanisms.