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.