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

Abstract

Background and purpose: Benzylidene-anabaseines (BAs) are partial agonists of the a7 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)-binding sites on muscle-type nAChRs. Key results: Both BAs non-competitively inhibited ACh activation of human fetal muscle nAChRs and sterically inhibited the specific binding of the NCAs [piperidyl-3,4- ]Nicotine competition-binding experiments confirmed that 4OH-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-binding 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.

Novel Positive Allosteric Modulators of the Human α7 Nicotinic Acetylcholine Receptor

“…Previous results indicate that barbiturates (noncompetitive antagonists) and benzylideneÀ anabaseine analogues (specific R7 AChR agonists) can interact with the resting Torpedo ion channel, finally increasing the affinity of crystal violet 39 and [ 3 H]TCP. 22 Because we did not see this pharmacological effect, the existence of a luminal site for these PAMs in AChRs is less plausible. Instead, a negative allosteric modulator site located apart from the ion channel (reviewed in refs 8 and 9) might account for the antagonistic action elicited by these compounds.…”

Pharmacological and molecular studies on the interaction of varenicline with different nicotinic acetylcholine receptor subtypes. Potential mechanism underlying partial agonism at human α4β2 and α3β4 subtypes

“…To determine whether varenicline interacts with the Torpedo and hα4β2 nAChR ion channels, additional studies were conducted using [ 3 H]TCP (20 nM) [21] and [ 3 H]imipramine (13 nM) [23]. The effect of varenicline on [ 3 H]cytisine, in the absence (nAChRs are in the resting but activatable state) and presence of 200 μM proadifen [24], and [ 3 H]TCP binding, in the presence of 1 mM CCh (nAChRs are mainly in the desensitized state), was also determined as previously described [21,23] After incubation (2 h), nAChR-bound radioligand was separated from free radioligand by a filtration assay [19][20][21][22][23]. The concentrationresponse data were curve-fitted by nonlinear least squares analysis using the Prism software (GraphPad Software, San Diego, CA).…”

“…To determine receptor selectivity, the effect of varenicline on [ 3 H] MLA (4.1 nM) binding to hα7 nAChRs, on [ 3 H]epibatidine (4.6 nM) binding to hα3β4 nAChRs, and on [ 3 H]cytisine (9.1 nM) binding to hα4β2, hα4β4, and Torpedo nAChRs, respectively, was studied as previously described [19][20][21][22]. To determine whether varenicline interacts with the Torpedo and hα4β2 nAChR ion channels, additional studies were conducted using [ 3 H]TCP (20 nM) [21] and [ 3 H]imipramine (13 nM) [23].…”

Insights Into Nicotinic Receptor Signaling in Nicotine Addiction: Implications for Prevention and Treatment

“…The nAChRs also can be activated by allosteric ligands binding to other transmembrane sites [ 121 , 122 ]. Some studies suggest that allosteric ligands bind to different regions and recognition sites other than those where acetylcholine binds and affect the function of nAChRs [ 26 , 64 , 69 , 77 , 114 , 115 , 119 , 123 - 126 ]. For example, five amino acids, located within the α-helical transmembrane domains TM1 (S222, A225), TM2 (M253), and TM4 (F455, C459), were identified as the binding sites of allosteric ligands of α7 nAChRs [ 127 ].…”