Use of an a3b4 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 a4 and other subunits, while off-target activity could be associated with ganglionic-type receptors containing a3b4 binding sites and other subunits, including b4, b2, a5, or a3 as a structural subunit in the pentamer. Additional interest in a3 b4 a5-containing receptors arises from genome-wide association studies linking these genes, and a single nucleotide polymorphism (SNP) in a5 in particular, to lung cancer and heavy smoking. While a3 and b4 readily form receptors in expression system such as the Xenopus oocyte, since a5 is not required for function, simple co-expression approaches may under-represent a5-containing receptors. We used a concatamer of human a3 and b4 subunits to form ligand-binding domains, and show that we can force the insertions of alternative structural subunits into the functional pentamers. These a3b4 variants differ in sensitivity to ACh, nicotine, varenicline, and cytisine. Our data indicated lower efficacy for varenicline and cytisine than expected for b4-containing receptors, based on previous studies of rodent receptors. We confirm that these therapeutically important a4 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 a5 SNP. However, our data validate this approach for further investigations. 2012 Elsevier Ltd. All rights reserved.