title: Additional acetylcholine (ACh) binding site at alpha4/alpha4 interface of (alpha4beta2)2alpha4 nicotinic receptor influences agonist sensitivity.
abstract: Nicotinic acetylcholine receptor (nAChR) α4 and β2 subunits assemble in two alternate stoichiometries to produce (α4β2)(2)α4 and (α4β2)(2)β2, which display different agonist sensitivities. Functionally relevant agonist binding sites are thought to be located at α4(+)/β2(-) subunit interfaces, but because these interfaces are present in both receptor isoforms, it is unlikely that they account for differences in agonist sensitivities. In contrast, incorporation of either α4 or β2 as auxiliary subunits produces isoform-specific α4(+)/α4(-) or β2(+)/β2(-) interfaces. Using fully concatenated (α4β2)(2)α4 nAChRs in conjunction with structural modeling, chimeric receptors, and functional mutagenesis, we have identified an additional site at the α4(+)/α4(-) interface that accounts for isoform-specific agonist sensitivity of the (α4β2)(2)α4 nAChR. The additional site resides in a region that also contains a potentiating Zn(2+) site but is engaged by agonists to contribute to receptor activation. By engineering α4 subunits to provide a free cysteine in loop C at the α4(+)α4(-) interface, we demonstrated that the acetylcholine responses of the mutated receptors are attenuated or enhanced, respectively, following treatment with the sulfhydryl reagent [2-(trimethylammonium)ethyl]methanethiosulfonate or aminoethyl methanethiosulfonate. The findings suggest that agonist occupation of the site at the α4(+)/(α4(-) interface leads to channel gating through a coupling mechanism involving loop C. Overall, we propose that the additional agonist site at the α4(+)/α4(-) interface, when occupied by agonist, contributes to receptor activation and that this additional contribution underlies the agonist sensitivity signature of (α4β2)(2)α4 nAChRs.
authors.affiliation: School of Life Sciences, Oxford Brookes University, Oxford OX3 0BP, United Kingdom.
authors.forename: Simone
authors.initials: S
authors.lastname: Mazzaferro
authors.affiliation: School of Life Sciences, Oxford Brookes University, Oxford OX3 0BP, United Kingdom.
authors.forename: Naïl
authors.initials: N
authors.lastname: Benallegue
authors.affiliation: Leibniz-Institut für Molekulare Pharmakologie and Neurocure Initiative Charité Universitäts Medizin, 13125 Berlin, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: Carbone
authors.affiliation: School of Life Sciences, Oxford Brookes University, Oxford OX3 0BP, United Kingdom.
authors.forename: Federica
authors.initials: F
authors.lastname: Gasparri
authors.affiliation: Structural Bioinformatics and Computational Biochemistry, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom.
authors.forename: Ranjit
authors.initials: R
authors.lastname: Vijayan
authors.affiliation: Structural Bioinformatics and Computational Biochemistry, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom.
authors.forename: Philip C
authors.initials: PC
authors.lastname: Biggin
authors.affiliation: Department of Pharmacology, University College London, London WC1E 6BT, United Kingdom.
authors.forename: Mirko
authors.initials: M
authors.lastname: Moroni
authors.affiliation: School of Life Sciences, Oxford Brookes University, Oxford OX3 0BP, United Kingdom. Electronic address: ibermudez@brookes.ac.uk.
authors.forename: Isabel
authors.initials: I
authors.lastname: Bermudez
mesh_terms: D000109:Acetylcholine / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004594:Electrophysiology / Q000379:methods; D006801:Humans; D007477:Ions / Q000737:chemistry; D016296:Mutagenesis; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering; D020033:Protein Isoforms; D011978:Receptors, Nicotinic / Q000378:metabolism*; D014982:Xenopus laevis; D015032:Zinc / Q000737:chemistry
chemical_list: D003432:Cross-Linking Reagents; D007477:Ions; D020033:Protein Isoforms; D011978:Receptors, Nicotinic; C403531:nicotinic receptor alpha4beta2; D015032:Zinc; D003545:Cysteine; D000109:Acetylcholine