title: Distinctive effects of nicotinic receptor intracellular-loop mutations associated with nocturnal frontal lobe epilepsy.
abstract: Previously characterized nicotinic acetylcholine receptor (nAChR) autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE)-associated mutations are found in α2, α4 and β2 subunit transmembrane (TM) domains. They predominantly increase ACh potency and, for β2-subunit mutants, increase macroscopic currents. Two recently-identified mutations, α4(R336H) and β2(V337G), located in the intracellular cytoplasmic loop (C2) have been associated with non-familial NFLE. Effects of these mutations on α4β2-nAChR function and expression were studied for the first time, using two-electrode voltage clamp recordings in Xenopus laevis oocytes. Biased-ratio preparations elucidated the mutations' effects at alternate isoforms: high-sensitivity [HS; (α4)2(β2)3] or low-sensitivity [LS; (α4)3(β2)2] via 1:10 or 30:1 [α4:β2] cRNA injection ratios, respectively. An unbiased (1:1 [α4:β2] cRNA) injection ratio was also used to study potential shifts in isoform expression. α4(R336H)-containing receptors showed significant increases in maximal ACh-induced currents (Imax) in all preparations (140% increase compared to wild type control). β2(V337G)-containing receptors significantly increased Imax in the LS-favoring preparation (20% increase compared to control). Expression of either mutation consistently produced enrichment of HS-isoform expression in all preparations. α4β2-nAChR harboring either NFLE mutant subunit showed unchanged ACh, sazetidine-A, nicotine, cytisine and mecamylamine potency. However, both mutant subunits enhanced partial agonist efficacies in the LS-biased preparation. Using β2-subunit-specific [(125)I]mAb 295 immunolabeling, nAChR cell-surface expression was determined. Antibody binding studies revealed that the β2(V337G) mutation tended to reduce cell-surface expression, and function per receptor was significantly increased by either NFLE mutant subunit in HS-favoring preparations. These findings identify both common and differing features between TM- and C2-domain AD/NFLE-associated mutations. As we discuss, the shared features may be particularly salient to AD/NFLE etiology.
authors.affiliation: Barrow Neurological Institute, Division of Neurobiology, 350 West Thomas Rd., Phoenix, AZ 85013, USA. Electronic address: maeganweltzin@gmail.com.
authors.forename: Maegan M
authors.initials: MM
authors.lastname: Weltzin
authors.affiliation: Department of Neuroscience, Perelman School of Medicine at the University of Pennsylvania, 217 Stemmler Hall, 36th and Hamilton Walk, Philadelphia, PA 19104-6074, USA. Electronic address: jslkk@mail.med.upenn.edu.
authors.forename: Jon M
authors.initials: JM
authors.lastname: Lindstrom
authors.affiliation: Barrow Neurological Institute, Division of Neurobiology, 350 West Thomas Rd., Phoenix, AZ 85013, USA. Electronic address: Ronald.Lukas@dignityhealth.org.
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Lukas
authors.affiliation: Barrow Neurological Institute, Division of Neurobiology, 350 West Thomas Rd., Phoenix, AZ 85013, USA. Electronic address: Paul.Whiteaker@dignityhealth.org.
authors.forename: Paul
authors.initials: P
authors.lastname: Whiteaker
mesh_terms: D000818:Animals; D017034:Epilepsy, Frontal Lobe / Q000235:genetics*; D009154:Mutation; D009865:Oocytes; D011978:Receptors, Nicotinic / Q000235:genetics*; D014981:Xenopus
chemical_list: D011978:Receptors, Nicotinic
keywords: (−)-nicotine hydrogen tartrate salt (PubChem CID: 89594); Acetylcholine chloride (PubChem CID: 6060); Cytoplasmic loop; Dihydro-β-erythroidine (PubChem CID: 31762); Nicotinic acetylcholine receptor; Nocturnal frontal lobe epilepsy; cytisine (PubChem CID: 10235); mecamylamine hydrochloride (PubChem CID: 13221); sazetidine-A (PubChem CID: 11983356); α4β2 isoform