Research Grants Program Office

Nicotinic acetylcholine receptors (nAChRs) play an important role in neurotransmission and are also involved in addiction and several disease states. There is significant interest in therapeutic targeting of nAChRs; however, achieving selectivity for one subtype over others has been a longstanding challenge, given the close structural similarities across the family. Here, we characterize binding interactions in the α3β4 nAChR subtype via structure-function studies involving noncanonical amino acid mutagenesis and two-electrode voltage clamp electrophysiology. We establish comprehensive binding models for both the endogenous neurotransmitter ACh and the smoking cessation drug cytisine. We also use a panel of C(10)-substituted cytisine derivatives to probe the effects of subtle changes in the ligand structure on binding. By comparing our results to those obtained for the well-studied α4β2 subtype, we identify several features of both the receptor and agonist structure that can be utilized to enhance selectivity for either α3β4 or α4β2. Finally, we characterize binding interactions of the α3β4-selective partial agonist AT-1001 to determine factors that contribute to its selectivity. These results shed new light on the design of selective nAChR-targeted ligands and can be used to inform the design of improved therapies with minimized off-target effects.