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 alpha 7 nicotinic acetylcholine receptor current by primarily increasing peak amplitude, a property of type I alpha 7 positive allosteric modulation. In this study, the effects of these two compounds were investigated at two different alpha 7/5-HT3 chimeras (chimera 1, comprising of extracellular alpha 7 N-terminus fused to the remainder of 5-HT3A, and chimera 2 containing an additional alpha 7 encoded M2-M3 loop), and wild-type alpha 7 and 5-HT3A 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)similar to 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 alpha 7 receptors. In chimera 2, genistein and 5-HI potentiated agonist-evoked responses (EC50: 4-5 mu M for genistein and 300-500 mu M for 5-HI) and at higher concentrations evoked current directly consistent with ago-allosteric modulation. At chimera 1 and 5-HT3A 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 alpha 7 agonist [H-3](1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo [2.2.1] heptane ([H-3]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 alpha 7 receptor modulatory sites offers unique opportunities for developing CNS therapeutics and understanding its pharmacology. (c) 2010 Elsevier B.V. All rights reserved.