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Published October 2015 | public
Journal Article Open

Heterologous expression and nonsense suppression provide insights into agonist behavior at α6β2 nicotinic acetylcholine receptors


The α6-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 α6-β2 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 α6β2 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-π interaction at several nAChR subtypes – and several agonists. These studies reveal that acetylcholine forms a strong cation-π interaction with the conserved tryptophan, while nicotine and TC299423 do not, suggesting a unique pharmacology for the α6β2 nAChR.

Additional Information

© 2015 Published by Elsevier Ltd. Received 14 January 2015; Received in revised form 27 March 2015; Accepted 10 April 2015; Available online 20 April 2015. We thank the NIH (NS 34407) for support of this work. MRP was supported by an NIH/NRSA training grant: 5 T32 GM07616.

Attached Files

Supplemental Material - mmc1.docx

Supplemental Material - mmc2.docx

Accepted Version - nihms683389.pdf


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August 22, 2023
August 22, 2023