Molecular Mechanism Of Calcium-activated Potassium Channel In The Activation Of Hainan Tarantula Toxin

The IK channel is a potassium ion channel that is bidirectionally controlled by both calcium ions and voltages.It is widely distributed in many types of cells,such as T lymphocytes,B lymphocytes,epithelial cells,fibroblasts,vascular endothelial cells,macrophages and Muscle cells etc.At the same time,IK channels are closely related to the regulation of cell physiological activities,such as cell number proliferation,cell migration,cells apoptosis and pyroptosis and programmed cell death controlled by genes.The current study found that IK channels are closely related to malignant tumors,airway remodeling,myocardial infarction,atherosclerosis,asthma,renal fibrosis,sickle cell disease and other diseases.Hainan tarantula-I（HNTX-I）is a high-abundance polypeptide isolated from Hainan tarantula with a relative molecular weight of 3608.1Da and contains 33 amino acid residues.The sequence is ECKGFGKSCVPGKNECCSGYACNSRDKWCKVLL.Although HNTX-I is the first peptide activator of the IK channel discovered to date,its activity is not ideal,and the mechanism of interaction between HNTX-I toxin and IK channels is also unclear.In order to solve the above problems,in-depth study of the molecular mechanism of HNTX-I toxin and IK channel interaction,HNTX-I and its 11 mutants were synthesized,these mutants are E1K,E1F,E1F-S18K,K3A,F5I,K7A,E15D,R25A,D26A,K27A,K30A.It was confirmed by patch-clamp experiments that the activity of the synthesized HNTX-I was consistent with the activity of the natural HNTX-I,indicating that the Post-synthesis renaturation peptideswas identical in structure to the natural HNTX-I.In a series of mutants of HNTX-I,K3A,F5I and K7A did not significantly activate IK channels at 40μM.The activation rate of IK channel by E1F,E1F-S18K and E1K are higher than that of HNTX-I at 40μM.E1F has the highest activity on IK channels.The activation rate of E1F at 4μM is20.1%±0.4%,the activation rate of E1F at 40μM is 78.5%±0.5%,and the activation rate of E1F at 80μM is 112.5%±0.7%.The EC₅₀0 of the Boltzmann sigmoidal equation fitting E1F for IK channel activation was8.01μM.The above results indicate that the amino acid residues at positions 1,3,5 and 7 on HNTX-I may be the key sites for the toxin to bind to the IK channel,and the toxin mainly utilizes the N-terminal amino acid to act on the IK channel.The most active HNTX-I mutant E1F was selected to study the binding mechanism of HNTX-I to IK channel.The IK current was significantly activated by the addition of 80μM E1F,and the addition of100 nM NS309 continued to activate the IK channel current approximately 1.25 times.The addition of 1μM TRAM-34 significantly inhibited the IK activated current.In order to further explore the binding mechanism between toxin and channel,a series of IK channel mutants were constructed by site-directed mutagenesis of alanine scanning of the extracellular regions of IK channels S1-S2,S3-S4 and S5-S6.At 40μM concentration,E1F had no obvious activation effect on IK channel mutants W141A and T260A,but the activation of G259A was significantly enhanced,indicating that these three loci regions are key locations for the interaction of toxin and channel.Combined with patch clamp results,the S1-S2 region of the mutant channel had no significant effect on the interaction of toxins and channels;the S3-S4 region and S5-S6 region of the mutant channel had a great influence on the IK channel,and the S5-S6 region Q229,V231,N232,T234,S238 etc.may form hydrophobic spots,the interaction activity between the toxin and the channel will decrease due to weakening of hydrophobic interaction after mutation.G259 near S6 may significantly enhance the affinity of E1F for IK channel due to weak hydrophobicity after mutation.On the contrary,T260 may weaken the interaction force due to the polarity enhancement after the mutation.The above conclusions indicate that the interaction of HNTX-I toxin with IK channel may be the result of action of hydrophobic.