Use of an α3β4 nicotinic acetylcholine receptor subunit concatamer to characterize ganglionic receptor subtypes with specific subunit composition reveals species-specific pharmacologic properties

Drug development for nicotinic acetylcholine receptors (nAChR) is challenged by subtype diversity arising from variations in subunit composition. On-target activity for neuronal heteromeric receptors is typically associated with CNS receptors that contain alpha 4 and other subunits, while off-target activity could be associated with ganglionic-type receptors containing alpha 3 beta 4 binding sites and other subunits, including beta 4, beta 2, alpha 5, or alpha 3 as a structural subunit in the pentamer. Additional interest in alpha 3 beta 4 alpha 5-containing receptors arises from genome-wide association studies linking these genes, and a single nucleotide polymorphism (SNP) in alpha 5 in particular, to lung cancer and heavy smoking. While alpha 3 and beta 4 readily form receptors in expression system such as the Xenopus oocyte, since alpha 5 is not required for function, simple co-expression approaches may under-represent alpha 5-containing receptors. We used a concatamer of human alpha 3 and beta 4 subunits to form ligand-binding domains, and show that we can force the insertions of alternative structural subunits into the functional pentamers. These alpha 3 beta 4 variants differ in sensitivity to ACh, nicotine, varenicline, and cytisine. Our data indicated lower efficacy for varenicline and cytisine than expected for beta 4-containing receptors, based on previous studies of rodent receptors. We confirm that these therapeutically important alpha 4 receptor partial agonists may present different autonomic-based side-effect profiles in humans than will be seen in rodent models, with varenicline being more potent for human than rat receptors and cytisine less potent. Our initial characterizations failed to find functional effects of the alpha 5 SNP. However, our data validate this approach for further investigations. (C) 2012 Elsevier Ltd. All rights reserved.