Photoaffinity Labeling the Agonist Binding Sites of Torpedo and α4β2 Nicotinic Acetylcholine Receptors and Acetylcholine Binding Proteins (AChBPs) with [3H]Cytisine

Abstract

The subunit composition of brain nicotinic acetylcholine receptors (nAChR) dictates their different physiological, pharmacological and pathophysiological properties. While targeting brain nAChRs is a promising strategy in the treatment of different neurological conditions including Alzheimer's and Parkinson's disease and nicotine dependence, the development of nAChR subtype-selective agents remains a challenge. The partial agonist cytisine and its derivative varenicline (CHANTIX; FDA approved drug for smoking cessation) are examples of drugs with higher selectivity for the α4β2 nAChR subtype. Here, we use radioligand binding, photoaffinity labeling and computational analysis to study the mode of interaction of cytisine with a diverse group of acetylcholine binding sites [nAChRs and ACh-binding proteins (AChBP)]. [3H]Cytisine binds with high affinity (1.6 nM) to α4β2 nAChRs, with low affinity (1.3 ∈μM) at both α-γ and α-δ agonist binding sites of the Torpedo (muscle-type) nAChR and to L-AChBP, A-AChBP and A-AChBP(Y55W) with low to modest affinity, 0.37 ∈μM, 2.5 ∈μM, and 80 nM, respectively. Upon UV irradiation, [3H]Cytisine photoincorporated selectively into the α- and γ-subunits of Torpedo nAChR . The sites of [3H]Cytisine labeling were determined in each subunit, αCys192/193,αTyr198 (Loop C), γTrp55 (Loop D) and γTyr117 (Loop E) of the agonist binding site. [3H]Cytisine efficiently photolabels the agonist binding sites of AChBPs and the α4β2 nAChR (both α4 and β2 subunits are labeled). The sites of [3H]Cytisine labeling in the Torpedo nAChR and in AChBPs and the α4β2 nAChR (ongoing experiments), along with results from cytisine docking simulations will be used to compare modes of interaction of α4β2 nAChR-selective and subtype non-selective agonists (e.g. ACh) to nAChRs and AChBPs.