title: Roles for N-terminal extracellular domains of nicotinic acetylcholine receptor (nAChR) β3 subunits in enhanced functional expression of mouse α6β2β3- and α6β4β3-nAChRs.
abstract: Functional heterologous expression of naturally expressed mouse α6*-nicotinic acetylcholine receptors (mα6*-nAChRs; where "*" indicates the presence of additional subunits) has been difficult. Here we expressed and characterized wild-type (WT), gain-of-function, chimeric, or gain-of-function chimeric nAChR subunits, sometimes as hybrid nAChRs containing both human (h) and mouse (m) subunits, in Xenopus oocytes. Hybrid mα6mβ4hβ3- (∼ 5-8-fold) or WT mα6mβ4mβ3-nAChRs (∼ 2-fold) yielded higher function than mα6mβ4-nAChRs. Function was not detected when mα6 and mβ2 subunits were expressed together or in the additional presence of hβ3 or mβ3 subunits. However, function emerged upon expression of mα6mβ2mβ3(V9'S)-nAChRs containing β3 subunits having gain-of-function V9'S (valine to serine at the 9'-position) mutations in transmembrane domain II and was further elevated 9-fold when hβ3(V9'S) subunits were substituted for mβ3(V9'S) subunits. Studies involving WT or gain-of-function chimeric mouse/human β3 subunits narrowed the search for domains that influence functional expression of mα6*-nAChRs. Using hβ3 subunits as templates for site-directed mutagenesis studies, substitution with mβ3 subunit residues in extracellular N-terminal domain loops "C" (Glu(221) and Phe(223)), "E" (Ser(144) and Ser(148)), and "β2-β3" (Gln(94) and Glu(101)) increased function of mα6mβ2*- (∼ 2-3-fold) or mα6mβ4* (∼ 2-4-fold)-nAChRs. EC50 values for nicotine acting at mα6mβ4*-nAChR were unaffected by β3 subunit residue substitutions in loop C or E. Thus, amino acid residues located in primary (loop C) or complementary (loops β2-β3 and E) interfaces of β3 subunits are some of the molecular impediments for functional expression of mα6mβ2β3- or mα6mβ4β3-nAChRs.
authors.affiliation: From the Department of Psychiatry and Neurobehavioral Sciences, School of Medicine, University of Virginia, Charlottesville, Virginia 22911 and.
authors.forename: Bhagirathi
authors.initials: B
authors.lastname: Dash
authors.affiliation: From the Department of Psychiatry and Neurobehavioral Sciences, School of Medicine, University of Virginia, Charlottesville, Virginia 22911 and.
authors.forename: Ming D
authors.initials: MD
authors.lastname: Li
authors.affiliation: Division of Neurobiology, Barrow Neurological Institute, Phoenix, Arizona 85013 Ronald.Lukas@DignityHealth.org.
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Lukas
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001285:Atropine / Q000378:metabolism / Q000494:pharmacology; D005260:Female; D015870:Gene Expression; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008969:Molecular Sequence Data; D009538:Nicotine / Q000378:metabolism / Q000494:pharmacology; D033661:Oocysts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis
chemical_list: D000596:Amino Acids; D021122:Protein Subunits; D011978:Receptors, Nicotinic; D011993:Recombinant Fusion Proteins; C568453:nicotinic receptor beta3, mouse; D009538:Nicotine; D001285:Atropine
keywords: Electrophysiology; Ion Channel; Neurotransmitter Receptor; Nicotinic Acetylcholine Receptors (nAChR); Receptor Structure-Function