The Electrophysiological Characteristics Of RBmK-AGAP And Mutants On Three Subtypes Of VGSCs And Validation In Vivo

Studies on the relationship of scorpion toxin and ion channels have demonstrated the invaluable prospects of scorpion toxins as molecular tools for dissecting the structure-function features of ion channels.It also makes scorpion toxins being promising drugs for treating ion channel-related diseases.However,scorpion toxins can kill organisms by inducing paralysis and arrhythmia,which results in their limitation in clinical application.Therefore,efforts need to be paid to the research on the relationship between the structure and bioactivity of scorpion toxins with the purpose to carry out further structural modification.In this paper,effects of mutants on hNav1.4,hNav1.5 and hNav1.7 were examined and compared with AGAP by using whole cell patch clamp.The analgesic activity of AGAP and mutants were further verified by thermal pain test and formalin-induced paw licking test.The data showed that D8K decreased the inhibition effects of peak currents of hNav1.4 and hNav1.5(IC50 values increased from 32.5 nM,21.9 nM to 234.4 nM,2.0 μM),while it showed similar analgesic activity with AGAP.W38F decreased the inhibition effects of peak currents of hNav1.4,hNav1.5 and hNav1.7(IC50 values increased from 32.5 nM,21.9 nM,14.1 nM to 38.0 mM,66.1μM,549.5 nM),while it reduced the analgesic activity compared with AGAP.W38G decreased the inhibition effects of peak currents of hNav1.4,hNav1.5 and hNav1.7(IC50 values increased from 32.5 nM,21.9 nM,14.1 nM to 2.7 mM,61.7 μM,177.8 nM),while it showed similar analgesic activity with AGAP.AGAP2 showed similar inhibition effects of peak currents of hNav1.4,hNav1.5 and hNav1.7,and analgesic activity.Due to the significantly decreased inhibition of hNav1.4 and hNav1.5,the toxic effects of W38G on skeletal and cardiac muscles were examined compared with AGAP.Correspondingly,W38G displayed much lower toxicities in skeletal and cardiac muscles than AGAP.In order to explore the possible mechanisms for analgesic activities,effects of AGAP and W38G were examined on the activity of hNav1.8.AGAP potently inhibited the activity of hNav1.8,indicating that both hNav1.7 and hNav1.8 were involved in analgesic mechanism of AGAP.Considering the similar analgesic effects,it seemed that the inhibition to hNav1.8 might take more responsibility for the analgesic effects of AGAP exhibited in rodent pain models induced by heat and noxious chemicals.In order to investigate whether double-peak concentration dependent phenomenon were also induced by inhibitors of VGSCs,several different VGSCs inhibitors(site1TTX,site2 safinamide,site4 AGAP,lidocaine and phenytoin sodium)were carried out.The data showed that all these inhibitors showed S-curve concentration dependent phenomenon on hNav1.4,hNav1.5 and hNav1.7 instead of double-peak.In conclusion,we demonstrated that rBmK-AGAP displayed as inhibitors of hNav1.4,hNav1.5,hNav1.7 and hNa,,1.8.rBmK-AGAP also exhibited potentially the functional properties of β-type scorpion toxin.The Asp8 and Trp38 were the key amino acids involved in bioactivity and biotoxicity of rBmK-AGAP.W38G might be a safer alternative for clinical application since it retained the analgesic efficacy with less toxicity to skeletal and cardiac muscles,while the decreased inhibition to hNav1.4 and hNav1.5 was supposed to be one major reason.Different VGSCs regulators showed different phenomenon on hNav1.4,hNav1.5 and hNav1.7.VGSCs activators displayed double-peak concentration dependent phenomenon on hNav1.4,hNav1.5 and hNav1.7.However,VGSCs inhibitors displayed typical S-curve concentration dependent phenomenon on hNav1.4,hNav1.5 and hNav1.7 instead of double-peak.