Peng_2013_Mol.Pharmacol_84_459

Positive allosteric modulators (PAMs) of alpha7 nicotinic acetylcholine receptors can enhance ion channel currents and downstream effects of alpha7 stimulation. We investigated the approach of using noncompetitive antagonists to regulate alpha7 receptor function, potentially distinguishing effects requiring ion channel currents from signaling induced by nonconducting states. Three small readily reversible antagonists, (1S,2R,4R)-N,2,3,3-tetramethylbicyclo[2.2.1]heptan-2-amine (mecamylamine), N-(2.6-dimethylphenylcarbamoylmethyl)triethylammonium bromide (QX-314), and 2-(dimethylamino)ethyl 4-(butylamino)benzoate (tetracaine), as well as three large slowly reversible antagonists, bis-(2,2,6,6-tetramethyl-4-piperidinyl) sebacate (BTMPS), 2,2,6,6-tetramethylpiperidin-4-yl heptanoate (TMPH), and 1,2,4,5-tetra-{5-[1-(3-benzyl)pyridinium]pent-1-yl}benzene tetrabromide (tkP3BzPB), were investigated for their effectiveness and voltage dependence in the inhibition of responses evoked by acetylcholine alone or augmented by the alpha7-selective PAM N-(5-chloro-2,4-dimethoxyphenyl)-N'-(5-methyl-3-isoxazolyl)-urea (PNU-120596). Analyses of the small antagonists on PNU-120596-potentiated single-channel bursts indicated that each agent had a distinct mechanism of inhibition and only that of QX-314 was consistent with simple open channel block. In addition to decreasing channel open times and burst durations, mecamylamine and tetracaine induced unique subconductance states. To determine whether channel-blocking activity alone would be sufficient to prevent cell death, the antagonists were tested for their ability to protect alpha7-expressing cells from cytotoxic effects of the alpha7 agonist choline in combination with PNU-120596. Only tetracaine and tkP3BzPB, the two agents that had effects least consistent with simple ion channel block, were fully cytoprotective at concentrations that gave submaximal inhibition of macroscopic currents in oocytes. Further analyses indicated that toxicity produced by PNU-120596 and choline was calcium independent and likely an apoptotic event. Our results are consistent with the hypothesis that PAMs may modulate conformational states important for both channel activity and ion channel-independent signaling.