title: Engineered α4β2 nicotinic acetylcholine receptors as models for measuring agonist binding and effect at the orthosteric low-affinity α4-α4 interface.
abstract: The nicotinic acetylcholine receptor α4β2 is important for normal mammalian brain function and is known to express in two different stoichiometries, (α4)2(β2)3 and (α4)3(β2)2. While these are similar in many aspects, the (α4)3(β2)2 stoichiometry differs by harboring a third orthosteric acetylcholine binding site located at the α4-α4 interface. Interestingly, the third binding site has, so far, only been documented using electrophysiological assays, actual binding affinities of nicotinic receptor ligands to this site are not known. The present study was therefore aimed at determining binding affinities of nicotinic ligands to the α4-α4 interface. Given that epibatidine shows large functional potency differences at α4-β2 vs. α4-α4 interfaces, biphasic binding properties would be expected at (α4)3(β2)2 receptors. However, standard saturation binding experiments with [(3)H]epibatidine did not reveal biphasic binding under the conditions utilized. Therefore, an engineered β2 construct (β2(HQT)), which converts the β(-) face to resemble that of an α4(-) face, was utilized to create (α4)3(β2(HQT))2 receptors harboring three α4-α4 interfaces. With this receptor, low affinity binding of epibatidine with a Kd of ∼5 nM was observed in sharp contrast to a Kd value of ∼10 pM observed for wild-type receptors. A strong correlation between binding affinities at the (α4)3(β2(HQT))2 receptor and functional potencies at the wild-type receptor of a range of nicotinic ligands highlighted the validity of using the mutational approach. Finally, large differences in activities at α4-β2 vs. α4-α4 interfaces were observed for structurally related agonists underscoring the need for establishing all binding parameters of compounds at α4β2 receptors.
authors.affiliation: Faculty of Pharmacy, The University of Sydney, NSW 2006, Australia; Saniona AB, 2750 Ballerup, Denmark.
authors.forename: Philip K
authors.initials: PK
authors.lastname: Ahring
authors.affiliation: Faculty of Pharmacy, The University of Sydney, NSW 2006, Australia; NeuroSearch A/S, 2750 Ballerup, Denmark; Faculty of Health and Medical Sciences, University of Copenhagen, 2100 Copenhagen, Denmark.
authors.forename: Jeppe A
authors.initials: JA
authors.lastname: Olsen
authors.affiliation: NeuroSearch A/S, 2750 Ballerup, Denmark.
authors.forename: Elsebet Ø
authors.initials: EØ
authors.lastname: Nielsen
authors.affiliation: DanPET AB, SE-216 19 Malmö, Sweden.
authors.forename: Dan
authors.initials: D
authors.lastname: Peters
authors.affiliation: Hevesy Laboratory, Technical University of Denmark, DTU Nutech, 4000 Roskilde, Denmark.
authors.forename: Martin H F
authors.initials: MH
authors.lastname: Pedersen
authors.affiliation: NeuroSearch A/S, 2750 Ballerup, Denmark; Faculty of Health and Medical Sciences, University of Copenhagen, 2100 Copenhagen, Denmark.
authors.forename: Line A
authors.initials: LA
authors.lastname: Rohde
authors.affiliation: Faculty of Health and Medical Sciences, University of Copenhagen, 2100 Copenhagen, Denmark.
authors.forename: Jette S
authors.initials: JS
authors.lastname: Kastrup
authors.affiliation: Faculty of Health and Medical Sciences, University of Copenhagen, 2100 Copenhagen, Denmark.
authors.forename: Azadeh
authors.initials: A
authors.lastname: Shahsavar
authors.affiliation: Faculty of Pharmacy, The University of Sydney, NSW 2006, Australia.
authors.forename: Dinesh C
authors.initials: DC
authors.lastname: Indurthi
authors.affiliation: Faculty of Pharmacy, The University of Sydney, NSW 2006, Australia.
authors.forename: Mary
authors.initials: M
authors.lastname: Chebib
authors.affiliation: Faculty of Health and Medical Sciences, University of Copenhagen, 2100 Copenhagen, Denmark. Electronic address: mig@sund.ku.dk.
authors.forename: Michael
authors.initials: M
authors.lastname: Gajhede
authors.affiliation: Faculty of Pharmacy, The University of Sydney, NSW 2006, Australia. Electronic address: thomas.balle@sydney.edu.au.
authors.forename: Thomas
authors.initials: T
authors.lastname: Balle
mesh_terms: D000109:Acetylcholine / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001381:Azepines / Q000493:pharmacokinetics; D001665:Binding Sites / Q000187:drug effects / Q000235:genetics; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000493:pharmacokinetics; D004305:Dose-Response Relationship, Drug; D004558:Electric Stimulation; D057809:HEK293 Cells; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics; D008958:Models, Molecular; D009538:Nicotine / Q000494:pharmacology; D018722:Nicotinic Agonists / Q000494:pharmacology*; D009865:Oocytes; D011485:Protein Binding / Q000187:drug effects; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D011725:Pyridines / Q000493:pharmacokinetics; D011978:Receptors, Nicotinic / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014316:Tritium / Q000493:pharmacokinetics; D014982:Xenopus laevis
chemical_list: C561587:1-(5-ethoxypyridin-3-yl)-1,4-diazepane; D001381:Azepines; D019086:Bridged Bicyclo Compounds, Heterocyclic; D018722:Nicotinic Agonists; D021122:Protein Subunits; D011725:Pyridines; D011978:Receptors, Nicotinic; C403531:nicotinic receptor alpha4beta2; D014316:Tritium; D009538:Nicotine; C082748:epibatidine; D000109:Acetylcholine
keywords: Cys-loop receptor; Electrophysiology; Ion channel; Nicotinic acetylcholine receptor (nAChR); Pharmacology; Radioligand binding