Expression,Purification And Electrophysiological Function Of The Recombinant Peptide ImKTx96 From The Venom Of The Scorpion Isometrus Maculates

The ion channels on the cell membrane are the basis of many important physiological activities in the organism.Changes in the number,structure,and function of ion channels often cause disease.Therefore,in recent years,ion channels are gradually becoming the target of many drug development.There are many types of ion channels,but in terms of the results found so far,potassium ion channels have the most subtypes and the most complex effects.It plays an important role in various physiological processes,such as regulating neuron excitability,hormone secretion,muscle contraction,neurotransmitter release,heart rate regulation,cell proliferation and cell volume regulation.The occurrence of potassium channel diseases is generally caused by mutations in genes encoding potassium channel or abnormal protein structure.Recent studies have shown that the KCNA 2 mutation encoding the voltage-gated potassium ion channel Kv1.2 is a new cause of epilepsy encephalopathy,so the Kv1.2 channel has become a potential new target for treating epilepsy encephalopathy.Scorpion toxin is the largest family of peptide potassium channel blockers.Since most scorpion toxins are small molecular peptides,they have the characteristics of short sequence,simple structure and strong functional specificity.In the past few decades,they have been successfully proved to be used as molecular probes to study channel structure and function,especially For Kv1.x,h ERG,SK,IK and BK channels.Therefore,finding scorpion toxins that specifically and selectively act on the Kv1.2 channel can lay a foundation for the treatment of epilepsy.First,in this study,a new potassium channel toxin,named ImKTx96,was screened from the venom gland c DNA library of the scorpion Isometrus maculates.Random clone sequencing analysis of the venom gland c DNA library revealed that the full length of ImKTx96 c DNA sequence was 306 nt,5’and 3’UTR were 24 nt and111 nt,respectively.A single‘aataaa’polyadenylation signal is found 18?nt upstream of the poly（A）tail in the 3’UTR end of the c DNA.The Open Reading Frame（ORF）of this c DNA sequence is 168 nt,encoding a total of 56 amino acid residues,including the signal peptide and the mature peptide,of which the signal peptide has27 amino acid residues and the mature peptide has 29 amino acid residues,and contains 3 pairs of disulfide bonds.Homologous sequence alignment revealed that ImKTx96 is 75%similar to the potassium channel scorpion toxinα-KTx10 subfamily toxins Co TX1 and Co TX2,and their cysteine positions are highly consistent,suggesting that they form CSαβmotif.Therefore,ImKTx96 is a new member of K⁺-channel scorpion toxinα-KTx10 group.Secondly,in this study,recombinant ImKTx96 peptide was expressed and purified in E.coli.In order to optimize expression in E.coli,primers for E.coli hobby codons were designed according to the amino acid sequence of ImKTx96 polypeptide,and the mature peptide coding gene of ImKTx96 was amplified by overlap PCR method,which was then digested by restriction endonucleases.The recombinant expression vector p GEX-6p-1-ImKTx96 was successfully constructed by ligase ligation,and finally the recombinant plasmid was transformed into E.coli Rosetta（DE3）.The GST-ImKTx96 fusion protein was obtained by IPTG induction expression,purified by GSH column affinity chromatography,then ultrafiltration desalted and concentrated,and the GST tag of the fusion protein was removed by enterokinase EK cleavage,and finally separated and purified by RP-HPLC to obtain chromatography rImKTx96 peptide,and its molecular weight was determined to be 3150.9 Da by MALDI-TOF-MS,which is consistent with theoretical calculations,indicating that the expressed polypeptide is the expected polypeptide.Finally,this study explored the electrophysiological activity of rImKTx96peptides on voltage-gated potassium channels.The patch clamp technique was used to determine the effect of rImKTx96 polypeptide on the voltage-gated potassium channel family.The experimental results showed that 10μM rImKTx96 had a strong inhibitory effect on h Kv1.2 channel（about 72%）,and its IC₅₀ value was 2.63±0.48μM.rImKTx96 has only a slight inhibitory effect on the current of the h Kv1.3channel（about 10%）,but basically has no effect on the other three subtypes of potassium ion channels h Kv1.1,h Kv3.2,and h Kv4.1.In order to study the mechanism of interaction between rImKTx96 and Kv1.2 channel,the 3D structure model of ImKTx96 was constructed by SWISS-MODEL molecular modeling,and the complex formed by the interaction of rImKTx96 and Kv1.2 ion channel was obtained by molecular docking.According to its model structural characteristics and electrophysiological function,ImKTx96 is one member of K⁺-channel scorpion toxinα-KTx10 group,and is also a good Kv1.2 channel inhibitor.In summary,this study cloned and identified a new potassium channel toxin gene ImKTx96 from the venom gland c DNA library of scorpion Isometrus maculates.The prokaryotic system was used to induce expression and purification to obtain highly purified recombined ImKTx96 peptide.The experiment confirmed that the recombined ImKTx96 peptide can effectively and specifically inhibit the current of the Kv1.2 ion channel.These research results may not only partially explain the toxicology of scorpion predation,but also found potential molecular probes to study the structure and function of the Kv1.2 ion channel,which laid a foundation for the study of scorpion toxins in epilepsy.