Welcome to the new version of CaltechAUTHORS. Login is currently restricted to library staff. If you notice any issues, please email coda@library.caltech.edu
Published January 28, 2014 | public
Journal Article

Structure Function Studies at Two Different Nicotinic Acetylcholine Receptor Subtypes

Abstract

The nicotinic acetylcholine receptor (nAChR) is involved with regulating the release of neurotransmitter in the synapse. This research focuses on two subtypes of neuronal nAChR, the α6β2 and α4β2 receptors. Subtypes containing the α6 and β2 subunits are found mainly in dopaminergic neurons. The acetylcholine binding site in these subtypes, which sits at the interface of the two subunits, is an important therapeutic target. Expression and activation of a pure population of α6β2 receptors in X. laevis oocytes has previously never been reported. This study used two mutations in the β2 M3-M4 loop and a reporter mutation in the M2 helix of each subunit to successfully express α6β2 receptors. Expression has been optimized, and a method to control subunit stoichiometry has been developed. We then used unnatural amino acid mutagenesis to develop a binding model for acetylcholine and nicotine at the α6-β2 subunit interface. The α4β2 subtype is the most prevalent nAChR subtype found in the brain and is implicated in many neurological disorders. Previous work has established a hydrogen bond interaction between the pyrrolidine nitrogen of nicotine and the carbonyl backbone adjacent to Trp149 in the α4 subunit. N'-methylnicotinium, which has a quaternary amine at the pyrrolidine nitrogen and cannot donate a hydrogen bond, was synthesized in order to probe this hydrogen bond donating interaction. Through a double-mutant cycle analysis utilizing an α-hydroxy acid mutation to perturb the carbonyl backbone hydrogen bond acceptor, we have determined that the hydrogen bonding interaction has a coupling energy of ∼2 kcal/mol.

Additional Information

© 2014 Biophysical Society. Published by Elsevier Inc.

Additional details

Created:
August 19, 2023
Modified:
October 26, 2023