title: New insight into active muscarinic receptors with the novel radioagonist [³H]iperoxo.
abstract: Activation of G protein-coupled receptors involves major conformational changes of the receptor protein ranging from the extracellular transmitter binding site to the intracellular G protein binding surface. GPCRs such as the muscarinic acetylcholine receptors are commonly probed with radioantagonists rather than radioagonists due to better physicochemical stability, higher affinity, and indifference towards receptor coupling states of the former. Here we introduce tritiated iperoxo, a superagonist at muscarinic M₂ receptors with very high affinity. In membrane suspensions of transfected CHO-cells, [³H]iperoxo - unlike the common radioagonists [³H]acetylcholine and [³H]oxotremorine M - allowed labelling of each of the five muscarinic receptor subtypes in radioagonist displacement and saturation binding studies. [³H]iperoxo revealed considerable differences in affinity between the even- and the odd-numbered muscarinic receptor subtypes with affinities for the M₂ and M₄ receptor in the picomolar range. Probing ternary complex formation on the M₂ receptor, [³H]iperoxo dissociation was not influenced by an archetypal allosteric inverse agonist, reflecting activation-related rearrangement of the extracellular loop region. At the inner side of M₂, the preferred Gi protein acted as a positive allosteric modulator of [³H]iperoxo binding, whereas Gs and Gq were neutral in spite of their robust coupling to the activated receptor. In intact CHO-hM₂ cells, endogenous guanylnucleotides promoted receptor/G protein-dissociation resulting in low-affinity agonist binding which, nevertheless, was still reported by [³H]iperoxo. Taken together, the muscarinic superagonist [³H]iperoxo is the best tool currently available for direct probing activation-related conformational transitions of muscarinic receptors.
authors.affiliation: Pharmacology & Toxicology Section, Institute of Pharmacy, University of Bonn, Gerhard-Domagk-Straße 3, 53121 Bonn, Germany.
authors.forename: Ramona
authors.initials: R
authors.lastname: Schrage
authors.affiliation: Pharmacology & Toxicology Section, Institute of Pharmacy, University of Bonn, Gerhard-Domagk-Straße 3, 53121 Bonn, Germany.
authors.forename: Janine
authors.initials: J
authors.lastname: Holze
authors.affiliation: Department of Pharmaceutical Chemistry, Institute of Pharmacy, University of Würzburg, Am Hubland, 97074 Würzburg, Germany.
authors.forename: Jessica
authors.initials: J
authors.lastname: Klöckner
authors.affiliation: Pharmacology & Toxicology Section, Institute of Pharmacy, University of Bonn, Gerhard-Domagk-Straße 3, 53121 Bonn, Germany.
authors.forename: Aileen
authors.initials: A
authors.lastname: Balkow
authors.affiliation: Pharmacology & Toxicology Section, Institute of Pharmacy, University of Bonn, Gerhard-Domagk-Straße 3, 53121 Bonn, Germany.
authors.forename: Anne S
authors.initials: AS
authors.lastname: Klause
authors.affiliation: Molecular-, Cellular-, and Pharmacobiology Section, Institute of Pharmaceutical Biology, University of Bonn, Nussallee 6, 53115 Bonn, Germany.
authors.forename: Anna-Lena
authors.initials: AL
authors.lastname: Schmitz
authors.affiliation: Dipartimento di Scienze Farmaceutiche, Sezione di Chimica Farmaceutica "Pietro Pratesi", Università degli Studi di Milano, via L. Mangiagalli 25, 20133 Milan, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: De Amici
authors.affiliation: Molecular-, Cellular-, and Pharmacobiology Section, Institute of Pharmaceutical Biology, University of Bonn, Nussallee 6, 53115 Bonn, Germany.
authors.forename: Evi
authors.initials: E
authors.lastname: Kostenis
authors.affiliation: Pharmacology & Toxicology Section, Institute of Pharmacy, University of Bonn, Gerhard-Domagk-Straße 3, 53121 Bonn, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Tränkle
authors.affiliation: Department of Pharmaceutical Chemistry, Institute of Pharmacy, University of Würzburg, Am Hubland, 97074 Würzburg, Germany.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Holzgrabe
authors.affiliation: Pharmacology & Toxicology Section, Institute of Pharmacy, University of Bonn, Gerhard-Domagk-Straße 3, 53121 Bonn, Germany. Electronic address: k.mohr@uni-bonn.de.
authors.forename: Klaus
authors.initials: K
authors.lastname: Mohr
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003412:Cricetulus; D054314:Drug Inverse Agonism; D004355:Drug Stability; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D006801:Humans; D007555:Isoxazoles / Q000819:agonists / Q000737:chemistry / Q000494:pharmacology*; D007700:Kinetics; D008024:Ligands; D008954:Models, Biological; D018721:Muscarinic Agonists / Q000737:chemistry / Q000494:pharmacology*; D019832:N-Methylscopolamine / Q000819:agonists / Q000737:chemistry / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D000644:Quaternary Ammonium Compounds / Q000819:agonists / Q000737:chemistry / Q000494:pharmacology*; D011869:Radioligand Assay; D043585:Receptor, Muscarinic M2 / Q000819:agonists / Q000235:genetics / Q000378:metabolism; D043588:Receptor, Muscarinic M4 / Q000819:agonists / Q000235:genetics / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014316:Tritium
chemical_list: C487078:CHRM2 protein, human; D007555:Isoxazoles; D008024:Ligands; D018721:Muscarinic Agonists; D000644:Quaternary Ammonium Compounds; D043585:Receptor, Muscarinic M2; D043588:Receptor, Muscarinic M4; D011976:Receptors, Muscarinic; D011994:Recombinant Proteins; C000591650:iperoxo; D014316:Tritium; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019832:N-Methylscopolamine
keywords: Acetylcholine; Allosteric modulation; Atropine; Cholera toxin (CTX); G protein-coupled receptor; Guanosine triphosphate (GTP); Iperoxo; Muscarinic acetylcholine receptor; N-methylscopolamine (NMS); Pertussis toxin (PTX); Pilocarpine; Superagonism; W84 (3-(1,3-dioxoisoindol-2-yl)propyl-[6-[3-(1,3-dioxoisoindol-2-yl)propyl-dimethylazaniumyl]hexyl]-dimethylazanium); [(3)H]iperoxo