title: Differential α4(+)/(-)β2 Agonist-binding Site Contributions to α4β2 Nicotinic Acetylcholine Receptor Function within and between Isoforms.
abstract: Two α4β2 nicotinic acetylcholine receptor (α4β2-nAChR) isoforms exist with (α4)2(β2)3 and (α4)3(β2)2 subunit stoichiometries and high versus low agonist sensitivities (HS and LS), respectively. Both isoforms contain a pair of α4(+)/(-)β2 agonist-binding sites. The LS isoform also contains a unique α4(+)/(-)α4 site with lower agonist affinity than the α4(+)/(-)β2 sites. However, the relative roles of the conserved α4(+)/(-)β2 agonist-binding sites in and between the isoforms have not been studied. We used a fully linked subunit concatemeric nAChR approach to express pure populations of HS or LS isoform α4β2*-nAChR. This approach also allowed us to mutate individual subunit interfaces, or combinations thereof, on each isoform background. We used this approach to systematically mutate a triplet of β2 subunit (-)-face E-loop residues to their non-conserved α4 subunit counterparts or vice versa (β2HQT and α4VFL, respectively). Mutant-nAChR constructs (and unmodified controls) were expressed in Xenopus oocytes. Acetylcholine concentration-response curves and maximum function were measured using two-electrode voltage clamp electrophysiology. Surface expression was measured with (125)I-mAb 295 binding and was used to define function/nAChR. If the α4(+)/(-)β2 sites contribute equally to function, making identical β2HQT substitutions at either site should produce similar functional outcomes. Instead, highly differential outcomes within the HS isoform, and between the two isoforms, were observed. In contrast, α4VFL mutation effects were very similar in all positions of both isoforms. Our results indicate that the identity of subunits neighboring the otherwise equivalent α4(+)/(-)β2 agonist sites modifies their contributions to nAChR activation and that E-loop residues are an important contributor to this neighbor effect.
authors.affiliation: From the Division of Neurobiology, Barrow Neurological Institute, Phoenix, Arizona 85013 and.
authors.forename: Linda M
authors.initials: LM
authors.lastname: Lucero
authors.affiliation: From the Division of Neurobiology, Barrow Neurological Institute, Phoenix, Arizona 85013 and.
authors.forename: Maegan M
authors.initials: MM
authors.lastname: Weltzin
authors.affiliation: From the Division of Neurobiology, Barrow Neurological Institute, Phoenix, Arizona 85013 and.
authors.forename: J Brek
authors.initials: JB
authors.lastname: Eaton
authors.affiliation: the Department of Neuroscience, University of Pennsylvania Medical School, Philadelphia, Pennsylvania 19104.
authors.forename: John F
authors.initials: JF
authors.lastname: Cooper
authors.affiliation: the Department of Neuroscience, University of Pennsylvania Medical School, Philadelphia, Pennsylvania 19104.
authors.forename: Jon M
authors.initials: JM
authors.lastname: Lindstrom
authors.affiliation: From the Division of Neurobiology, Barrow Neurological Institute, Phoenix, Arizona 85013 and.
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Lukas
authors.affiliation: From the Division of Neurobiology, Barrow Neurological Institute, Phoenix, Arizona 85013 and paul.whiteaker@dignityhealth.org.
authors.forename: Paul
authors.initials: P
authors.lastname: Whiteaker
mesh_terms: D000109:Acetylcholine / Q000737:chemistry; D000495:Allosteric Site; D000818:Animals; D001384:Azetidines / Q000737:chemistry; D001665:Binding Sites / Q000235:genetics; D018076:DNA, Complementary / Q000378:metabolism; D004594:Electrophysiology; D005786:Gene Expression Regulation; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009538:Nicotine / Q000737:chemistry; D018722:Nicotinic Agonists / Q000737:chemistry; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D020033:Protein Isoforms; D012313:RNA / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000737:chemistry*; D014982:Xenopus laevis
chemical_list: D000109:Acetylcholine; D001384:Azetidines; D018076:DNA, Complementary; D009538:Nicotine; D018722:Nicotinic Agonists; D020033:Protein Isoforms; D012313:RNA; D011978:Receptors, Nicotinic; C403531:nicotinic receptor alpha4beta2; C082735:azetidine
keywords: electrophysiology; ligand-gated ion channels; monoclonal antibody; nicotinic acetylcholine receptors (nAChR); receptor structure-function; site-directed mutagenesis