Marotta_2014_Neuropharmacol_77_342

Nicotinic acetylcholine receptors (nAChRs) containing the alpha5 subunit are of interest because genome-wide association studies and candidate gene studies have identified polymorphisms in the alpha5 gene that are linked to an increased risk for nicotine dependence, lung cancer, and/or alcohol addiction. To probe the functional impact of an alpha5 subunit on nAChRs, a method to prepare a homogeneous population of alpha5-containing receptors must be developed. Here we use a gain of function (9') mutation to isolate populations of alpha5-containing nAChRs for characterization by electrophysiology. We find that the alpha5 subunit modulates nAChR rectification when co-assembled with alpha4 and beta2 subunits. We also probe the alpha5-alpha4 interface for possible ligand-binding interactions. We find that mutations expected to ablate an agonist-binding site involving the alpha5 subunit have no impact on receptor function. The most straightforward interpretation of this observation is that agonists do not bind at the alpha5-alpha4 interface, in contrast to what has recently been demonstrated for the alpha4-alpha4 interface in related receptors. In addition, our mutational results suggest that the alpha5 subunit does not replace the alpha4 or beta2 subunits and is relegated to occupying only the auxiliary position of the pentameric receptor.