Nicotinic acetylcholine receptors (AChRs) are multimeric proteins assembled from a wide variety of subunits. Each combination of subunits has unique pharmacological and physiological properties, resulting in many different receptor subtypes that could serve different functional roles. Agonists such as acetylcholine activate the channel by binding to at least two subunits in the receptor. All of the different subunits have a unique disulfide bond near the ACh binding site, and when this disulfide is reduced, nicotinic receptor function is largely blocked. The applicant proposes to use a novel set of labeling and cross-linking agents based on arsenical chemistry in order to analyze the subunit composition of receptors using subunit-specific antibodies. These agents are designed to potently and covalently attach to the reduced disulfide. The applicant proposes to make radioiodinated and biotinylated arsenical ligands, show that they label the Torpedo AChR (as a model), and then test them on neuronal receptors in the chick nervous system. In addition, a series of photoactivatable crosslinking ligands will also be used to determine the subunits adjacent to the subunits. The applicant will also use a series of biotinylated arsenical reagents to map the distance from the disulfide bond in the binding site from the surface of the receptor.
| Lee, H K; Gwalani, L; Mishra, V et al. (2009) Investigating the role of protein folding and assembly in cell-type dependent expression of alpha7 nicotinic receptors using a green fluorescent protein chimera. Brain Res 1259:7-16 |
