Stokes_2012_Neuropharmacol_63_538

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 alpha4 and other subunits, while off-target activity could be associated with ganglionic-type receptors containing alpha3beta4 binding sites and other subunits, including beta4, beta2, alpha5, or alpha3 as a structural subunit in the pentamer. Additional interest in alpha3 beta4 alpha5-containing receptors arises from genome-wide association studies linking these genes, and a single nucleotide polymorphism (SNP) in alpha5 in particular, to lung cancer and heavy smoking. While alpha3 and beta4 readily form receptors in expression system such as the Xenopus oocyte, since alpha5 is not required for function, simple co-expression approaches may under-represent alpha5-containing receptors. We used a concatamer of human alpha3 and beta4 subunits to form ligand-binding domains, and show that we can force the insertions of alternative structural subunits into the functional pentamers. These alpha3beta4 variants differ in sensitivity to ACh, nicotine, varenicline, and cytisine. Our data indicated lower efficacy for varenicline and cytisine than expected for beta4-containing receptors, based on previous studies of rodent receptors. We confirm that these therapeutically important alpha4 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 alpha5 SNP. However, our data validate this approach for further investigations.