Symmetrical Bispyridinium Compounds Act as Open Channel Blockers of Cation-Selective Ion Channels.

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Current treatments against organophosphate poisoning (OPP) do not directly address effects mediated by the overstimulation of nicotinic acetylcholine receptors (nAChR). Non-oxime bispyridinium compounds (BPC) promote acetylcholine esterase-independent recovery of organophosphate-induced paralysis. Here, we test the hypothesis that they act by positive modulatory action on nAChRs. Using two-electrode voltage clamp analysis in combination with mutagenesis and molecular docking analysis, the potency and molecular mode of action of a series of nine BPCs was investigated on human α7 and muscle-type nAChRs expressed in Xenopus laevis oocytes. The investigated BPCs inhibited α7 and/or muscle-type nAChRs with IC50 values in the high nanomolar to high micromolar range. Further analysis of the most potent analogues revealed a noncompetitive, voltage-dependent inhibition. Co-application with the α7-selective positive allosteric modulator PNU120596 and generation of α7/5HT3 receptor chimeras excluded direct interaction with the PNU120596 binding site and binding to the extracellular domain of the α7 nAChR, suggesting that they act as open channel blockers (OCBs). Molecular docking supported by mutagenesis localized the BPC binding area in the outer channel vestibule between the extracellular and transmembrane domains. Analysis of BPC action on other cation-selective channels suggests a rather nonspecific inhibition of pentameric cation channels. BPCs have been shown to ameliorate organophosphate-induced paralysis in vitro and in vivo. Our data support molecular action as OCBs at α7 and muscle-type nAChRs and suggest that their positive physiological effects are more complex than anticipated and require further investigation.