Gronlien_2010_Eur.J.Pharmacol_647_37

Genistein and 5-hydroxyindole (5-HI) potentiate the alpha7 nicotinic acetylcholine receptor current by primarily increasing peak amplitude, a property of type I alpha7 positive allosteric modulation. In this study, the effects of these two compounds were investigated at two different alpha7/5-HT(3) chimeras (chimera 1, comprising of extracellular alpha7 N-terminus fused to the remainder of 5-HT(3A), and chimera 2 containing an additional alpha7 encoded M2-M3 loop), and wild-type alpha7 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 alpha7 receptors. In chimera 2, genistein and 5-HI potentiated agonist-evoked responses (EC(50): 4-5 muM for genistein and 300-500 muM 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 alpha7 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 alpha7 receptor modulatory sites offers unique opportunities for developing CNS therapeutics and understanding its pharmacology.