Heterologous expression and nonsense suppression provide insights into agonist behavior at ?6?2 nicotinic acetylcholine receptors.

ABSTRACT: The ?6-containing subtypes of the nicotinic acetylcholine receptor (nAChR) are localized to presynaptic terminals of the dopaminergic pathways of the central nervous system. Selective ligands for these nAChRs are potentially useful in both Parkinson's disease and addiction. For these and other goals, it is important to distinguish the binding behavior of agonists at the ?6-?2 binding site versus other subtypes. To study this problem, we apply nonsense suppression-based non-canonical amino acid mutagenesis. We report a combination of four mutations in ?6?2 that yield high-level heterologous expression in Xenopus oocytes. By varying mRNA injection ratios, two populations were observed with unique characteristics, likely due to differing stoichiometries. Responses to nine known nAChR agonists were analyzed at the receptor, and their corresponding EC50 values and efficacies are reported. The system is compatible with nonsense suppression, allowing structure-function studies between Trp149 - a conserved residue on loop B found to make a cation-? interaction at several nAChR subtypes - and several agonists. These studies reveal that acetylcholine forms a strong cation-? interaction with the conserved tryptophan, while nicotine and TC299423 do not, suggesting a unique pharmacology for the ?6?2 nAChR.

SUBMITTER: Post MR 

PROVIDER: S-EPMC4635625 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature