Abstract Neuronal nicotinic acetylcholine receptors are essential therapeutic targets for addiction, mental health and neurodegenerative disorders. These pentameric ligand-gated ion channels are the prototypical members of the Cys-loop receptor superfamily, which mediate fast neurotransmission throughout the central and peripheral nervous systems. Here we propose to determine high-resolution structures of two representative neuronal nicotinic receptor subtypes. Structural analysis of nicotinic receptors has been hampered by the challenges of recombinant expression of eukaryotic membrane proteins. These proteins typically express at low levels and are unstable after purification. Furthermore, most nicotinic receptors are obligate heteromers and in many cases these heteromers can assemble as pentamers with different ratios of subunits. This complication of mixed stoichiometry is present among all Cys-loop receptor families but is best characterized in the nicotinic receptors. The structural heterogeneity results in physiologically important, finely tuned pharmacological and channel properties. In Aim 1, we propose to develop methods for expression and purification of these receptors that can assemble in multiple stoichiometries. In Aims 2 and 3 we propose to apply these approaches to determine structures of two heteromeric nicotinic receptors in physiologically- relevant and functionally-distinct alternate stoichiometries. The individual receptor structures will provide key insights into the structural underpinnings of ion permeation and ligand recognition. Comparison of the structures of the two receptors, and of alternative stoichiometries of the same receptor, will provide a reliable structural foundation for understanding the distinctive biophysical and pharmacological properties of each receptor subunit combination.

Narrative This research application is focused on the structure and function of nicotinic acetylcholine receptors, which are neurotransmitter receptors critical for regulating central and peripheral nervous system function. These proteins play key roles in addiction, in particular to nicotine, and in neurodegenerative disease and mental illness. The proposed studies will elucidate how both neurotransmitters and addictive drugs interact with this important class of cell surface receptors, providing essential information for improved therapeutics targeting these receptors.