ABSTRACT Nicotinic acetylcholine receptors are ubiquitous in the brain and underlie regulation of attention and arousal by cholinergic neurons. nAChRs are highly expressed in neurons of the reinforcement and reward pathways, and maladaptive effects on nAChRs resulting from drug exposure contribute to drug addiction. These receptors are crucial in addiction both to nicotine (a direct orthosteric agonist of nAChRs) and to other drugs of abuse resulting from drug induced plastic changes in cholinergic activity in reward centers. A persistent challenge facing any nAChR-targeted pharmaceutical approach against addiction is the broad diversity in assembly and distribution of the brain-expressed forms of the protein. The long-term goal of this project is to develop an accurate and comprehensive methodology to identify and therapeutically target the full family of native nAChR conformations. The short-term goal of this R03 proposal is to demonstrate that a yeast-display nanobody library, screened against two distinct, widely expressed and physiologically critical conformations of nAChRs, will demonstrate the ability to robustly parse two separate stoichiometries of the nAChR in in vitro fluorescence and electrophysiology assays. The rationale is to deliver proof-of-concept that this approach can specifically target individual receptor types, to stimulate further characterization of successfully identified reagents for their possible functionality in modifying nAChR activity, and ultimately to establish the approach as a feasible method to study the native expression and assembly properties of the entire family of native nAChRs. This study includes two related objectives: 1) to develop a nanobody library enriched for yeast-mounted variable domain modules that potently bind purified 42 nAChRs and 2) to establish an effective counter-screening assay to deplete stoichiometrically non-specific nanobodies to purify interactors that selectively target two physiologically relevant, structurally distinct assemblies of 42. This represents an innovative application of the cutting-edge yeast nanobody screening method against a persistent roadblock in understanding the maladaptive changes to nicotinic receptor protein expression and distribution. Development and delivery of a biologic toolbox to study and manipulate all native nAChRs in vivo, as is the overall goal of this research, would represent a significant biomedical advance to investigate and combat addiction and other human ailments associated with the cholinergic nervous system.