title: Rare human nicotinic acetylcholine receptor α4 subunit (CHRNA4) variants affect expression and function of high-affinity nicotinic acetylcholine receptors.
abstract: Nicotine, the primary psychoactive component in tobacco smoke, produces its behavioral effects through interactions with neuronal nicotinic acetylcholine receptors (nAChRs). α4β2 nAChRs are the most abundant in mammalian brain, and converging evidence shows that this subtype mediates the rewarding and reinforcing effects of nicotine. A number of rare variants in the CHRNA4 gene that encode the α4 nAChR subunit have been identified in human subjects and appear to be underrepresented in a cohort of smokers. We compared three of these variants (α4R336C, α4P451L, and α4R487Q) to the common variant to determine their effects on α4β2 nAChR pharmacology. We examined [(3)H]epibatidine binding, interacting proteins, and phosphorylation of the α4 nAChR subunit with liquid chromatography and tandem mass spectrometry (LC-MS/MS) in HEK 293 cells and voltage-clamp electrophysiology in Xenopus laevis oocytes. We observed significant effects of the α4 variants on nAChR expression, subcellular distribution, and sensitivity to nicotine-induced receptor upregulation. Proteomic analysis of immunopurified α4β2 nAChRs incorporating the rare variants identified considerable differences in the intracellular interactomes due to these single amino acid substitutions. Electrophysiological characterization in X. laevis oocytes revealed alterations in the functional parameters of activation by nAChR agonists conferred by these α4 rare variants, as well as shifts in receptor function after incubation with nicotine. Taken together, these experiments suggest that genetic variation at CHRNA4 alters the assembly and expression of human α4β2 nAChRs, resulting in receptors that are more sensitive to nicotine exposure than those assembled with the common α4 variant. The changes in nAChR pharmacology could contribute to differences in responses to smoked nicotine in individuals harboring these rare variants.
authors.affiliation: Department of Psychiatry, Yale University School of Medicine, New Haven, Connecticut (T.D.M.-B., A.D.L., J.G., M.R.P.); Institute for Behavioral Genetics, University of Colorado, Boulder, Boulder, Colorado (T.D.M.-B.); Department of Pharmacology and Therapeutics, University of Florida, Gainesville, Florida (R.L.P., C.S.); W.M. Keck Biotechnology Research Laboratory (K.S.), Interdepartmental Neuroscience Program (A.D.L., M.R.P.), Department of Genetics (J.G., P.X.), and Department of Neurobiology, Yale University School of Medicine, New Haven, Connecticut (M.R.P.); Department of Psychiatry, Veterans Affairs Connecticut Healthcare Center, West Haven, Connecticut (J.G.); Center for Human Genome Variation, Duke University, Durham, North Carolina (P.X.); and Department of Neuroscience, Medical School of the University of Pennsylvania, Philadelphia, Pennsylvania (J.L.).
authors.forename: T D
authors.initials: TD
authors.lastname: McClure-Begley
authors.forename: R L
authors.initials: RL
authors.lastname: Papke
authors.forename: K L
authors.initials: KL
authors.lastname: Stone
authors.forename: C
authors.initials: C
authors.lastname: Stokes
authors.forename: A D
authors.initials: AD
authors.lastname: Levy
authors.forename: J
authors.initials: J
authors.lastname: Gelernter
authors.forename: P
authors.initials: P
authors.lastname: Xie
authors.forename: J
authors.initials: J
authors.lastname: Lindstrom
authors.forename: M R
authors.initials: MR
authors.lastname: Picciotto
mesh_terms: D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D009538:Nicotine / Q000494:pharmacology; D018722:Nicotinic Agonists / Q000494:pharmacology; D009865:Oocytes / Q000502:physiology; D010766:Phosphorylation; D011110:Polymorphism, Genetic; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D011978:Receptors, Nicotinic / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation; D014982:Xenopus laevis
chemical_list: D019086:Bridged Bicyclo Compounds, Heterocyclic; D009538:Nicotine; D018722:Nicotinic Agonists; D021122:Protein Subunits; D011725:Pyridines; D011978:Receptors, Nicotinic; C403531:nicotinic receptor alpha4beta2; C082748:epibatidine