title: Importance of M2-M3 loop in governing properties of genistein at the α7 nicotinic acetylcholine receptor inferred from α7/5-HT3A chimera.
abstract: Genistein and 5-hydroxyindole (5-HI) potentiate the α7 nicotinic acetylcholine receptor current by primarily increasing peak amplitude, a property of type I α7 positive allosteric modulation. In this study, the effects of these two compounds were investigated at two different α7/5-HT(3) chimeras (chimera 1, comprising of extracellular α7 N-terminus fused to the remainder of 5-HT(3A), and chimera 2 containing an additional α7 encoded M2-M3 loop), and wild-type α7 and 5-HT(3A) receptors. Agonist-evoked responses, examined by expression of the chimeras in Xenopus laevis oocytes or HEK-293 cells, revealed that currents decayed slower and compounds {rank order: N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-282987)~2-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5-phenyl-1,3,4-oxadiazole (NS6784)>acetylcholine>choline} were more potent in chimera 2 than chimera 1 or α7 receptors. In chimera 2, genistein and 5-HI potentiated agonist-evoked responses (EC(50): 4-5 μM for genistein and 300-500 μM for 5-HI) and at higher concentrations evoked current directly consistent with ago-allosteric modulation. At chimera 1 and 5-HT(3A) receptors, neither compound directly evoked any current and 5-HI, only at chimera 1, was able to potentiate agonist-evoked responses. Genistein and 5-HI did not inhibit the binding of the α7 agonist [(3)H](1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1] heptane ([(3)H]A-585539) to rat brain or chimera 2. In summary, this study supports the role of the M2-M3 loop being critical for the positive allosteric effect of genistein, but not 5-HI, and in agonist-evoked response fine-tuning. The identification of distinct α7 receptor modulatory sites offers unique opportunities for developing CNS therapeutics and understanding its pharmacology.
authors.affiliation: Neuroscience Research, Global Pharmaceutical Research and Development, Abbott, Oslo, Norway.
authors.forename: Jens Halvard
authors.initials: JH
authors.lastname: Grønlien
authors.forename: Hilde
authors.initials: H
authors.lastname: Ween
authors.forename: Kirsten
authors.initials: K
authors.lastname: Thorin-Hagene
authors.forename: Steven
authors.initials: S
authors.lastname: Cassar
authors.forename: Jinhe
authors.initials: J
authors.lastname: Li
authors.forename: Clark A
authors.initials: CA
authors.lastname: Briggs
authors.forename: Murali
authors.initials: M
authors.lastname: Gopalakrishnan
authors.forename: John
authors.initials: J
authors.lastname: Malysz
mesh_terms: D000109:Acetylcholine / Q000378:metabolism / Q000494:pharmacology; D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000378:metabolism / Q000494:pharmacology*; D005260:Female; D019833:Genistein / Q000378:metabolism / Q000494:pharmacology*; D057809:HEK293 Cells; D006801:Humans; D007211:Indoles / Q000378:metabolism / Q000494:pharmacology*; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D018722:Nicotinic Agonists / Q000378:metabolism / Q000494:pharmacology*; D009865:Oocytes / Q000187:drug effects; D017434:Protein Structure, Tertiary; D011724:Pyridazines / Q000378:metabolism / Q000494:pharmacology*; D051381:Rats; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000187:drug effects*; D011993:Recombinant Fusion Proteins; D012701:Serotonin / Q000378:metabolism / Q000494:pharmacology; D014982:Xenopus laevis; D064569:alpha7 Nicotinic Acetylcholine Receptor
chemical_list: C525870:2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo(2.2.1)heptane; D019086:Bridged Bicyclo Compounds, Heterocyclic; C579167:Chrna7 protein, human; C579165:Chrna7 protein, rat; D007211:Indoles; D018722:Nicotinic Agonists; D011724:Pyridazines; D011978:Receptors, Nicotinic; D011993:Recombinant Fusion Proteins; D064569:alpha7 Nicotinic Acetylcholine Receptor; C026785:5-hydroxyindole; D012701:Serotonin; D019833:Genistein; D000109:Acetylcholine