Transcriptome Analysis And Functional Identification Of Four Potassium Toxins From Scorpiops_pococki In Tibet

The potassium ion channel is a kind of transmembrane protein that mediates the transport of potassium ions on the cell membrane,which is widely distributed among various tissues and organs of the human body.It is mainly divided into inward rectifying potassium channel,double-hole potassium channel,voltage-gated potassium channel and calcium-activated potassium channel.Besides,these channels also play an important regulatory role in physiological and pathological processes of the human body.Many scorpion toxin peptides can specifically act on potassium channels,and the typical one is an alpha-potassium toxin,which plays an important role in the study of the relationship between the structure and function of potassium channels.The alpha-potassium toxins in scorpion toxins are usually composed of20⁴0 amino acid residues and contain 3⁴ pairs of disulfide bonds.Four alpha-potassium toxins,KTX-SP1,KTX-SP2,KTX-SP3,and KTX-SP5,were selected from the cDNA library of Scorpiops_pococki in Tibet in this study to screen for regulatory peptides that can specifically act on Kv1.3 channel related to autoimmune diseases.Through the methods of genetic engineering,whole-cell patched clamp technology,calcium ion fluorescence imaging technology and ELISA detection of inflammatory factors,we obtained Kv1.3 channel inhibitor KTX-SP2 that can regulate the autoimmune response,providing a new lead active molecule for the development of drugs to treat autoimmune diseases.Firstly,the transcriptome analysis and expression vector construction of Scorpiops_pococki in Tibet were carried out in this paper.In this study,the total RNA of Scorpiops_pococki from Tibet were extracted,the transcriptome sequence results were analyzed and functional annotation was performed.Four scorpion toxin polypeptides,KTX-SP1,KTX-SP2,KTX-SP3,and KTX-SP5,were screened out,and recombinant plasmids of toxin polypeptides were constructed using pGEX-4T-1as vector plasmids.Secondly,the recombinant expression and functional identification of scorpion toxin polypeptides were carried out.The chromatographic pure toxin polypeptides were prepared by GST fusion protein expression in E.coli prokaryotic expression system.By mass spectrometry,the actual measured value of the toxin polypeptide molecule was basically consistent with the theoretical value.The function of alpha-potassium toxin KTX-SP1,KTX-SP2,KTX-SP3,and KTX-SP5,were identified by the whole-cell patched clamp technique.KTX-SP3 and KTX-SP5 with 3?M were found to have no significant inhibitory effect on Kv1.1,Kv1.2,and Kv1.3 channel currents.Among them,1?M KTX-SP1 can inhibit about 60%of Kv1.3 channel currents,which activity is about 700nM,but it has no significant inhibition of Kv1.1and Kv1.2 channel current.The same concentration of KTX-SP2 can inhibit about 70%of Kv1.1 channel current and 80%of Kv1.2 channel current,respectively,and the300nM KTX-SP2 almost completely inhibits the Kv1.3 channel current.This led to the discovery of a novel scorpion toxin polypeptide that specifically acts on Kv1.3channel,providing a material basis for the study on the mechanism of scorpion toxin's interaction with Kv1.3 channel.Finally,KTX-SP2 was used as a molecular probe to identify its function and the further investigating of the immunomodulatory mechanism of KTX-SP2 on Jurkat T cells was carried out.The IC₅₀ values of KTX-SP2 for Kv1.1,Kv1.2,and Kv1.3channels were determined by whole-cell patched clamp technique to be484.99±25.5nM,56.896±2.32 nM and 14.72±1.98nM,respectively.In addition,its activity on Kv1.3 channel of Jurkat T cells was 29.094±1.12 nM.However,KTX-SP2has no significant inhibitory effect on sodium channel current,which indicates that KTX-SP2 has a selective inhibitory effect on potassium channel.In the autoimmune response,the activation of T cells is dependent on Ca²⁺signaling and the release of inflammatory factor IL-2.Therefore,this study demonstrated that KTX-SP2 could inhibit the release of free Ca²⁺signal and inflammatory factor IL-2 in Jurkat T cells by blocking Kv1.3 channel through calcium ion imaging technology and ELISA assay,indicating the correct direction for further study on the structure and function of KTX-SP2.To sum up,this paper carried out the expression purification and functional identification of a series of polypeptides of Scorpiops_pococki toxin from Tibet and screened for the alpha-potassium toxin KTX-SP2 that specifically acted on the Kv1.3channel.Taking KTX-SP2 as the molecular basis,we studied the interaction mechanism between KTX-SP2 and potassium ion channel and conducted in-depth research on the pharmacological activity of KTX-SP2 regulating the autoimmune response by inhibiting Kv1.3 channel,which greatly promoted the development and application of scorpion toxin in the field of biomedicine.