Study On The Molecular Mechanism Of Scorpion Toxin On The Mutation Of Sodium Channel In Insects

Voltage-gated sodium channel is a kind of complete membrane protein and the channel of sodium ion selectively passing through the biofilm.It plays an important role in excitable cells and affects the physiological and pathological process of the whole organism.There are nine different isomers of sodium channels in mammals,all of which have very similar characteristics,but have obvious differences in pharmacological functions.Sodium channels are also targets of many drugs,including scorpion toxin peptides that act on sodium channels.According to the action mode of scorpion toxin on sodium channel and the difference of binding site,scorpion toxin can be divided intoα-scorpion toxin andβ-scorpion toxin.α-scorpion toxin mainly binds to site 3 on sodium channel to delay the inactivation of sodium channel.β-scorpion toxin binds to site 4 of sodium channel and can change the gating characteristics of sodium channel.LqhαIT belongs toα-scorpion toxin,which has a significant effect on insect sodium channels but not on mammals.In this study,aiming at this type ofα-scorpion toxin,the sodium channel of Blattella germanica was mutated by site-directed mutation.Five different types of sodium channel single mutants（V295N,M293A,K1676T,D1672A,D1672R）were constructed and heterogeneously expressed in Xenopus oocytes.The sodium current was detected by double-electrode voltage clamp detection system.The results showed that all the five sodium channel mutants were successfully expressed within 2-3 days,and the peak sodium current was more than 2μA,and then each mutant was stimulated with scorpion venom.The EC₅₀ values of V295N,M293A and K1676T mutants were4267.44,189.89 and 253.33n M,respectively.The V295N mutant reduced the sensitivity ofα-scorpion toxin to sodium channel by 21 times,while the mutants M293A and K1676T had little effect on the binding ofα-scorpion toxin.D1672A/R mutants can greatly reduce the sensitivity ofαscorpion toxin to sodium channels.α-scorpion toxin is a kind of synthetic polypeptide toxin.in this experiment,different types of mutants of insect sodium channel and different types ofα-scorpion toxin were constructed,which were F17G,K8D and E15F,respectively.The sensitivity of insect sodium channel was tested.The EC₅₀ value of wild typeα-scorpion toxin was 914.96n M,F17G and 9191.98 n M.However,K8D could not bind to sodium channel at the concentration of 1000n M,and its sensitivity was much lower than that of wild typeα-scorpion toxin.The EC₅₀ value of E15F was 339.17,which was 3 times higher than that of wild type,indicating that theα-scorpion toxin of E15F mutant could bind to insect sodium channels more easily.In addition,among the five Blattella germanica mutants,only the D1672A/R mutant shifted the voltage-dependent sodium channel to depolarization 8m V in the activated state,but had no effect on the fast inactivation of the channel.Finally,the three-dimensional models of sodium channels of wild type and V295N mutants were constructed by molecular modeling.Through the comparison of three-dimensional models,it is found that the V295N mutation does not directly affect the binding of sodium channel toα-scorpion toxin,but leads to the decrease of the binding efficiency ofα-scorpion toxin to sodium channel by changing the conformation of sodium channel.This result better shows that the change of amino acid residues not in the binding region will also lead to the decrease ofα-scorpion toxin binding efficiency,and provides a new idea for the development of new agents in the future.