George_2012_J.Biol.Chem_287_25151

Genome-wide studies have strongly associated a non-synonymous polymorphism (rs16969968) that changes the 398th amino acid in the nAChR alpha5 subunit from aspartic acid to asparagine (D398N), with greater risk for increased nicotine consumption. We have used a pentameric concatemer approach to express defined and consistent populations of alpha3beta4alpha5 nAChR in Xenopus oocytes. alpha5(Asn-398; risk) variant incorporation reduces ACh-evoked function compared with inclusion of the common alpha5(Asp-398) variant without altering agonist or antagonist potencies. Unlinked alpha3, beta4, and alpha5 subunits assemble to form a uniform nAChR population with pharmacological properties matching those of concatemeric alpha3beta4* nAChRs. alpha5 subunit incorporation reduces alpha3beta4* nAChR function after coinjection with unlinked alpha3 and beta4 subunits but increases that of alpha3beta4alpha5 versus alpha3beta4-only concatemers. alpha5 subunit incorporation into alpha3beta4* nAChR also alters the relative efficacies of competitive agonists and changes the potency of the non-competitive antagonist mecamylamine. Additional observations indicated that in the absence of alpha5 subunits, free alpha3 and beta4 subunits form at least two further subtypes. The pharmacological profiles of these free subunit alpha3beta4-only subtypes are dissimilar both to each other and to those of alpha3beta4alpha5 nAChR. The alpha5 variant-induced change in alpha3beta4alpha5 nAChR function may underlie some of the phenotypic changes associated with this polymorphism.