Molecular actions of smoking cessation drugs at α4β2 nicotinic receptors defined in crystal structures of a homologous binding protein Bert Billena, Radovan Spurnya, Marijke Bramsa, René van Elkb, Soledad Valera-Kummerc, Jerrel L. Yakeld, Thomas Voetse, Daniel Bertrandc, August B. Smitb, and Chris Ulensa,1 aLaboratory of Structural Neurobiology, KU Leuven, 3000 Leuven, Belgium; bDepartment of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, VU University, 1081 HV, Amsterdam, The Netherlands; cHiQScreen, 1211 Geneva, Switzerland; dLaboratory of Neurobiology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 22709; and eLaboratory of Ion Channel Research, KU Leuven, 3000 Leuven, Belgium Edited by Palmer Taylor, University of California at San Diego, La Jolla, CA, and accepted by the Editorial Board April 26, 2012 (received for review October 5, 2011) Partial agonists of the α4β2 nicotinic acetylcholine receptor domain in complex with α-bungarotoxin (16). However, signifi- (nAChR), such as varenicline, are therapeutically used in smoking cant progress has been made since the discovery of water-soluble cessation treatment. These drugs derive their therapeutic effect pentameric acetylcholine binding proteins (AChBPs) from snails from fundamental molecular actions, which are to desensitize and the subsequent elucidation of their high-resolution ligand- α4β2 nAChRs and induce channel opening with higher affinity, bound crystal structures. AChBPs are homologs of the extracel- but lower efficacy than a full agonist at equal receptor occupancy. lular domain of nAChRs and are the best-studied structural Here, we report X-ray crystal structures of a unique acetylcholine models of ligand recognition by the extracellular domain of binding protein (AChBP) from the annelid Capitella teleta, Ct- nAChRs (17–19). AChBP, in complex with varenicline or lobeline, which are both In this study, we report the high-resolution X-ray crystal partial agonists. These structures highlight the architecture for structures of an AChBP from the marine worm Capitella teleta molecular recognition of these ligands, indicating the contact res- (20), a unique nonmolluscan AChBP, in complex with two partial PHARMACOLOGY idues that potentially mediate their molecular actions in α4β2 agonists, lobeline and the smoking cessation aid varenicline. nAChRs. We then used structure-guided mutagenesis and electro- These structures offer detailed insight into high-affinity inter- physiological recordings to pinpoint crucial interactions of vareni- actions of lobeline and varenicline in the binding pocket of Ct- cline with residues on the complementary face of the binding site AChBP. We then used structure-guided mutagenesis and elec- in α4β2 nAChRs. We observe that residues in loops D and E are trophysiological recordings of α4β2 nAChRs to reveal the key molecular determinants of desensitization and channel opening interactions of varenicline with the complementary face of the with limited efficacy by the partial agonist varenicline. Together, binding pocket that account for receptor desensitization and this study analyzes molecular recognition of smoking cessation channel opening with limited efficacy. By defining structural drugs by nAChRs in a structural context. determinants of these molecular actions at the α4β2 nAChR, we open avenues for the rational design of smoking cessation aids. addiction | cys-loop receptor | ligand-gated ion channel Results obacco smoking is a major cause of premature death world- Pharmacological Characterization of Ct-AChBP. In this study, we Twide. To a large extent, the reinforcing effects of nicotine result characterized the pharmacological properties of an AChBP from from the direct activation of neuronal α4β2 nicotinic acetylcholine the marine worm Capitella teleta, which is a unique nonmolluskan receptors (nAChRs), which triggers downstream events such as AChBP (20). To investigate the validity of Ct-AChBP as a poten- increased dopamine release in the mesolimbic system (1–4). tial model for the nAChR, we compared the binding properties of Therefore, the α4β2 nAChR subtype has become a key target for varenicline and various other ligands with two well-described development of therapeutic agents for smoking cessation (5, 6). In molluskan AChBPs, namely AChBP from Aplysia californica (Ac- particular, high-affinity partial agonists for α4β2 nAChRs, such as AChBP) and Lymnaea stagnalis (Ls-AChBP). Using competitive 3 lobeline (7), cytisine (8), and varenicline (8), are molecules of in- binding assays with H-epibatidine, we calculated Ki values for terest because they exhibit the unique property of acting as a mixed these ligands (Table 1). When Ct-AChBP is compared with the agonist/antagonist. High-affinity varenicline binding competes other AChBPs, we conclude that it has a distinct pharmacological fi fi with nicotine at the α4β2 nAChR and, thereby, antagonizes the pro le. Ct-AChBP shows a low af nity for acetylcholine and a high fi fi reward sensation of smoking (9, 10). However, varenicline acti- af nity for nicotine. The af nity of varenicline for Ct-AChBP is vates α4β2 nAChRs with low efficacy and desensitizes, leaving more than 10-fold higher than for Ac-AChBP and is close to the channels unopened even at high binding occupancy, thereby minimizing withdrawal symptoms and increasing the success rate of smoking cessation attempts (9, 10). Understanding the molec- Author contributions: B.B., R.v.E., S.V.-K., D.B., A.B.S., and C.U. designed research; B.B., R.S., M.B., R.v.E., S.V.-K., D.B., and C.U. performed research; M.B. and J.L.Y. contributed ular mechanism of partial agonism at the nAChR has great po- new reagents/analytic tools; B.B., R.S., M.B., R.v.E., S.V.-K., T.V., D.B., A.B.S., and C.U. tential for designing novel smoking cessation compounds but, as analyzed data; and B.B., M.B., R.v.E., T.V., D.B., A.B.S., and C.U. wrote the paper. yet, is hampered by the absence of the high-resolution structure of The authors declare no conflict of interest. a eukaryote nicotinic receptor. This article is a PNAS Direct Submission. P.T. is a guest editor invited by the Editorial Board. Current structural insight is derived from medium-resolution Freely available online through the PNAS open access option. electron microscopy images of the nAChR from Torpedo mar- Data deposition: The structural coordinates and structure factors reported in this paper have morata (11), X-ray structures from prokaryotic nAChR homologs been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 4AFG and 4AFH). GLIC (12, 13) and ELIC (14), the Caenorhabditis elegans gluta- 1To whom correspondence should be addressed. E-mail: [email protected]. mate-activated chloride channel GluCl (15) and an X-ray struc- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ture from the monomeric mouse nAChR α-subunit extracellular 1073/pnas.1116397109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1116397109 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 Table 1. Binding properties of Ct-AChBP in comparison with Ac-AChBP, Ls-AChBP, and α4β2 nAChRs Ligand Ct-AChBP Ac-AChBP Ls-AChBP α4β2 nAChR Ligand action Lobeline 0.29 ± 0.06 1.2 ± 0.4 1,000 ± 64 7 ± 1 Partial agonist Epibatidine 4.6 ± 0.3 11.4 ± 0.7 2.5 ± 0.2 0.05 ± 0.01 Agonist Varenicline 5 ± 172± 83± 1 0.18 ± 0.01 Partial agonist Strychnine 6 ± 138± 3223± 26 819 ± 90 Antagonist d-tubocurarine 140 ± 6509± 38 171 ± 18 2 ± 1 Antagonist Cytisine 247 ± 31 206 ± 52 219 ± 66 0.9 ± 0.1 Partial agonist Methyllycaconitine 358 ± 66 15 ± 415± 6 ND Antagonist Nicotine 423 ± 59 583 ± 84 1,110 ± 231 6 ± 2 Partial agonist α-conotoxin ImI > 1mM 4± 2 > 1 mM ND Antagonist Levamisole 4.6 ± 0.8 μM 6.0 ± 0.4 μM27± 3 μM ND Agonist Acetylcholine 57 ± 6 μM35± 5 μM29± 3 μM28± 4 μM Agonist Serotonine 171 ± 76 μM319± 143 μM893± 364 μM8± 3 μM Antagonist GABA > 1mM > 1mM > 1mM —— All measurements are in nanomolars unless otherwise noted. Binding constants (Ki values) were determined by using a competitive binding assay with 3H-epibatidine (n ≥ 3). ND, not determined. high affinity for Ls-AChBP. The high affinity of lobeline and transmembrane domain in integral Cys-loop receptors. The Cys varenicline for Ct-AChBP resembles the high-affinity binding to loop in Ct-AChBP comprises only 8 amino acids (C137–C146), α4β2 nAChR (7 ± 1 nM for lobeline and 0.18 ± 0.01 nM for var- compared with 12 amino acids in molluskan AChBPs and 13 in enicline; Table 1). Lobeline and varenicline both display partial membrane integral Cys-loop receptors. The β1-β2 loop in Ct- agonist action at α4β2 nAChR (7, 9). The pharmacology of α4β2 AChBP is 4 amino acids longer than in Ac-AChBP (M53–N56). nAChRs is complex because these receptors can occur in alternate Finally, Ct-AChBP has different conformations in the β5 strand, stoichiometries and contain ligand binding sites with high and low which lines the pentamer vestibule, and loop F, which forms part sensitivity (21). In addition, these receptors can occur in different of the ligand binding site. Although subtle differences exist, these allosteric conformations and display cooperativity upon ligand binding, which are properties lacking in AChBPs. Despite these limitations, our binding data demonstrate high-affinity binding of lobeline and varenicline and suggest that Ct-AChBP is a useful addition to the family of AChBPs as a suitable model for structural studies. X-Ray Crystal Structures of Ct-AChBP in Complex with Lobeline and Varenicline. The lobeline-bound structure of Ct-AChBP (1.9 Å, crystallographic details in Table S1) is shown along the fivefold symmetry axis with the C terminus of the protein pointing toward the viewer in Fig. 1A and away from the viewer in Fig.