Function of Human α3β4α5 Nicotinic Acetylcholine Receptors Is Reduced by the α5(D398N) Variant

Genome-wide studies have strongly associated a non-synonymous polymorphism (rs16969968) that changes the 398th amino acid in the nAChR alpha 5 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 alpha 3 beta 4 alpha 5 nAChR in Xenopus oocytes. alpha 5(Asn-398; risk) variant incorporation reduces ACh-evoked function compared with inclusion of the common alpha 5(Asp-398) variant without altering agonist or antagonist potencies. Unlinked alpha 3, beta 4, and alpha 5 subunits assemble to form a uniform nAChR population with pharmacological properties matching those of concatemeric alpha 3 beta 4* nAChRs. alpha 5 subunit incorporation reduces alpha 3 beta 4* nAChR function after coinjection with unlinked alpha 3 and beta 4 subunits but increases that of alpha 3 beta 4 alpha 5 versus alpha 3 beta 4-only concatemers. alpha 5 subunit incorporation into alpha 3 beta 4* 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 alpha 5 subunits, free alpha 3 and beta 4 subunits form at least two further subtypes. The pharmacological profiles of these free subunit alpha 3 beta 4-only subtypes are dissimilar both to each other and to those of alpha 3 beta 4 alpha 5 nAChR. The alpha 5 variant-induced change in alpha 3 beta 4 alpha 5 nAChR function may underlie some of the phenotypic changes associated with this polymorphism.