Importance of M2-M3 loop in governing properties of genistein at the α7 nicotinic acetylcholine receptor inferred from α7/5-HT3A chimera.
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.
Grønlien
Jens Halvard
JH
Neuroscience Research, Global Pharmaceutical Research and Development, Abbott, Oslo, Norway.
Ween
Hilde
H
Thorin-Hagene
Kirsten
K
Cassar
Steven
S
Li
Jinhe
J
Briggs
Clark A
CA
Gopalakrishnan
Murali
M
Malysz
John
J
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
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