title: Probing the non-canonical interface for agonist interaction with an α5 containing nicotinic acetylcholine receptor.
abstract: Nicotinic acetylcholine receptors (nAChRs) containing the α5 subunit are of interest because genome-wide association studies and candidate gene studies have identified polymorphisms in the α5 gene that are linked to an increased risk for nicotine dependence, lung cancer, and/or alcohol addiction. To probe the functional impact of an α5 subunit on nAChRs, a method to prepare a homogeneous population of α5-containing receptors must be developed. Here we use a gain of function (9') mutation to isolate populations of α5-containing nAChRs for characterization by electrophysiology. We find that the α5 subunit modulates nAChR rectification when co-assembled with α4 and β2 subunits. We also probe the α5-α4 interface for possible ligand-binding interactions. We find that mutations expected to ablate an agonist-binding site involving the α5 subunit have no impact on receptor function. The most straightforward interpretation of this observation is that agonists do not bind at the α5-α4 interface, in contrast to what has recently been demonstrated for the α4-α4 interface in related receptors. In addition, our mutational results suggest that the α5 subunit does not replace the α4 or β2 subunits and is relegated to occupying only the auxiliary position of the pentameric receptor.
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 E California Blvd., Pasadena, CA 91125, USA.
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Marotta
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 E California Blvd., Pasadena, CA 91125, USA.
authors.forename: Crystal N
authors.initials: CN
authors.lastname: Dilworth
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA.
authors.forename: Henry A
authors.initials: HA
authors.lastname: Lester
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 E California Blvd., Pasadena, CA 91125, USA. Electronic address: dadougherty@caltech.edu.
authors.forename: Dennis A
authors.initials: DA
authors.lastname: Dougherty
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000502:physiology*; D009538:Nicotine / Q000378:metabolism*; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D009538:Nicotine; D021122:Protein Subunits; D011978:Receptors, Nicotinic; C523972:CHRNA5 protein, mouse
keywords: A2B3; A3B2; Addiction; Biophysics; CNS; Cholinergic receptor; Electrophysiology; Hill coefficient; Ion channels; Nicotinic acetylcholine receptors; SEM; central nervous system; high sensitivity (α4β2)(2)(β2) receptor; low sensitivity (α4β2)(2)(α4) receptor; nAChR; nH; nicotinic acetylcholine receptor; standard error of the mean